Ex vivo antigen-presenting cells or activated CD-positive T cells for treatment of cancer

ABSTRACT

This disclosure is directed to methods of preparing dendritic cells or other CD40 bearing antigen-presenting cells and methods of treating cancer by using the dendritic cells or other antigen-presenting cells in combination with anti-chemorepellant agents. This disclosure is further directed to methods of preparing T cells and methods of treating cancer, by activated T cells optionally in combination with anti-chemorepellant agents. The antigen presenting cells of the disclosure are activated by incubation with cancer cells and fusion proteins. The T cells of the disclosures are activated by incubation with activated antigen-presenting cells that were activated by incubation with cancer cells and a fusion protein. In particular, the fusion protein comprises an antigen-binding domain, e.g., an antibody or antibody fragment, and a stress protein domain.

STATEMENT OF PRIORITY

This application is a 35 U.S.C. § 371 atonal phase application of PCTApplication PCT/US2017/050625 filed Sep. 8, 2017, which claims thebenefit of U.S. Provisional Application Ser. No. 62/385,880, filed Sep.9, 2016 and U.S. Provisional Application Ser. No. 62/385,898, filed Sep.9, 2016, the entire contents of each of which are incorporated byreference herein in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support. The U.S. Government hascertain rights in this invention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing, in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 1417-17_ST25.txt, 16,074 bytes in size, generated onMar. 7, 2019 and filed via EFS-Web, is provided in lieu of a paper copy.This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

This disclosure is directed to methods of preparing dendritic cells orother CD40 bearing antigen-presenting cells and methods of treatingcancer by using the dendritic cells or other antigen-presenting cells incombination with anti-chemorepellant agents. This disclosure is furtherdirected to methods of preparing T cells and methods of treating cancerby using activated T cells optionally in combination withanti-chemorepellant agents. The antigen presenting cells of thedisclosure are activated by incubation with cancer cells and fusionproteins. The T cells of the disclosures are activated by incubationwith activated antigen-presenting cells that were activated byincubation with cancer cells and a fusion protein. In particular, thefusion protein comprises an antigen-binding domain, e.g., an antibody orantibody fragment, and a stress protein domain.

BACKGROUND OF THE INVENTION

Conventional treatments for cancers, e.g., surgery, radiation, andchemotherapy, are often accompanied by substantial side effects, cancerrelapses, and physical suffering. For example, chemotherapies, which areintended to kill aggressively dividing cancer cells, may alsononspecifically target normal cells that are rapidly proliferating,e.g., the cells in blood, intestinal tract, and hair. This leads tosevere side effects, e.g., anemia, diarrhea, fatigue, and hair loss.

Immunotherapy is a promising treatment for cancers as well as otherdiseases. The immunotherapy treats disease by inducing, enhancing,augmenting, or suppressing an immune response in a patient. Usingimmunotherapy, the immune system can be stimulated to recognize cancercells, thereby creating long-lasting therapeutic effects against cancerrecurrence or relapse. Moreover, by focusing on enhancing the immunesystem rather than targeting proliferating cells, immunotherapies aremore specific and cause fewer side effects compared to conventionalcancer treatments.

Therefore, there is a need for a more effective immunotherapy to promotean immune response against cancer.

SUMMARY OF THE INVENTION

Immunotherapy is becoming an increasingly important tool in thearmamentarium against cancers. However, efficacy of immunotherapy hasnot been robust as was hoped, due in part to reduced antigenicity of theantigens and the subsequently weakened immunological response. Onepotential solution is to increase a patient's own immune response todisease-associated antigens by contacting T cells withantigen-presenting cells that express tumor-specific immunogenicantigens on the cell surface. Either the antigen-presenting cells or theresulting activated T cells may be administered to the patient.

Stress proteins are very efficient at activating antigen-presentingcells to provoke a T cell response. They have been particularlyeffective at eliciting cell mediated immune and humoral immune responsesby this pathway.

In one aspect, the present invention is directed to a method forpreparing activated antigen-presenting cells (“APCs”) by incubatingimmune cells ex vivo in the presence of tumor cells (or othertumor-specific antigen(s)) and a fusion protein for a time periodsufficient to produce the activated APCs.

In another aspect, the present invention is directed to a method forpreparing tumor-specific T cells via activated APCs by incubating immunecells ex vivo in the presence of tumor cells (or other tumor-specificantigen(s)) and a fusion protein for a time period sufficient to producethe activated APCs, optionally isolating the APCs from the tumorcells/antigens and fusion protein, then incubating the T cells andactivated APCs for a time period sufficient to produce activated,tumor-specific T cells, particularly CD3-positive T cells.

The fusion protein binds to antigens with high affinity, is highlyimmunogenic, exhibits MHC class I priming, provokes a T cell response,and is able to be produced in non-mammalian systems such as E. coli. Thefusion protein is thus suitable for use as a highly immunogenic vaccinefor the prevention or treatment of infectious, inflammatory, autoimmune,or malignant disease. The fusion protein may also act as a potentialcandidate presented to APCs to ultimately provoke T cell responses andincrease the specificity of immunotherapy against tumors. Non-limitingexamples of fusion proteins can be found in U.S. Pat. Nos. 8,143,387 and7,943,133; U.S. Patent Pub. No. 20110129484; and PCT Application NumberPCT/US2017/021911; each of which is incorporated herein by reference inits entirety.

Some tumor cells secrete concentrations of chemokines that aresufficient to repel immune cells from the site of a tumor, thus creatinga “chemorepellant wall” or “fugetactic wall” around the tumor. Thechemorepellant wall reduces the immune system's ability to target anderadicate the tumor. For example, repulsion of tumor antigen-specific Tcells, e.g., from a tumor expressing high levels of CXCL12 orinterleukin 8 (IL-8), allows the tumor cells to evade immune control.Anti-chemorepellant agents may inhibit the chemorepellant activity oftumor cells and allow the patient's immune system to target the tumor.Anti-chemorepellant agents and the systemic delivery ofanti-chemorepellant agents are known in the art (see, for example, U.S.Patent Application Publication No. 2008/0300165, incorporated herein byreference in its entirety).

Without being bound by theory, it is believed that the antigen-bindingdomain of the fusion protein will bind to the target (e.g., cancer cellsor tumor), and the stress protein domain will induce maturation ofantigen-presenting cells (e.g., dendritic cells). In combination, theanti-chemorepellant agent will inhibit the chemorepellant activity ofthe cancer cells with regard to the antigen-presenting cells and/or Tcells, such that the immune cells are able to penetrate the“chemorepellant wall” and access the target. In some embodiments, thecombination results in additive or synergistic effects.

In one aspect, this invention relates to methods of preparing activatedAPCs, which comprises incubating immune cells ex vivo in the presence ofcancer or tumor cells and a fusion protein for a time period sufficientto produce the activated APCs, wherein at least one activated APCdisplays an antigen derived from the cancer or tumor cells. In oneembodiment, the method further comprises isolating the activated APCs,wherein the isolated activated APCs are free or substantially free ofthe cancer or tumor cells and/or the fusion protein.

In one aspect, this invention relates to methods of preparing activatedT cells, which comprises incubating T cells with activated APCs for aperiod of time sufficient to produce activated T cells, wherein the APCswere prepared by incubating immune cells ex vivo in the presence ofcancer or tumor cells and a fusion protein for a time period sufficientto produce the activated APCs, wherein at least one activated APCdisplays an antigen derived from the cancer or tumor cells. In oneembodiment, the method further comprises isolating the activated Tcells. In one embodiment, the isolated activated T cells are free orsubstantially free of the activated APCs, cancer or tumor cells and/orthe fusion protein. In one embodiment, the APCs comprise dendriticcells.

In one aspect, the fusion protein comprises an antigen-binding componentand a heat shock protein component. While the antigen-binding componentbinds to the tumor cells, the heat shock protein component activates theAPCs. In one embodiment, the antigen-binding component of the fusionprotein may be any antibody or other molecule that recognizes a tumor orcancer cell of interest. In one embodiment, the antigen-bindingcomponent is a single chain antibody, a variable domain, or a fragmentantigen-binding (“Fab”) domain of an antibody.

In one aspect, the tumor cells are obtained from a patient.

In one aspect, the antigen-binding binding component is specific for acancer antigen (e.g., a tumor-specific antigen or a tumor-associatedantigen). The cancer antigen may be any identifiable cell surfaceantigen that is expressed by a cancer of interest. In one embodiment,the cancer antigen is mesothelin, alphafetoprotein, CEA, CA-125, MUC-1,Her2Neu, ETA, NY-ESO-1, VEGF, VEGFR1, VEGFR2, PSMA, prostate specificantigen, HPV17E7, mutant p53, surviving, ras, MAGE, gp100, tyrosinase,WT1, PR1, folate-binding protein, CA-19-9, FAP, G250, or A33. In oneembodiment, the antibody is specific for mesothelin. Fusion proteinsthat recognize and bind to mesothelin are described, for example, inU.S. Pat. No. 7,943,133, which is incorporated herein by reference inits entirety. Additional fusion proteins are described in PCTApplication No. PCT/US2017/021911, which is incorporated herein byreference in its entirety.

In one embodiment, the antigen-presenting cells include, but are notlimited to, dendritic cells, B lymphocytes, and mononuclear phagocytes.The mononuclear phagocytes may include monocytes and/or macrophages. Insome aspects, the APCs comprise at least one CD40. In another aspect,the APCs are dendritic cells.

In one embodiment, the heat shock protein component includes but is notlimited to HSP70 and/or an immune activating fragment and/or modifiedsequence thereof. In another aspect, the HSP70 or the immune activatingfragment and/or modified sequence thereof is from Mycobacteriumtuberculosis.

In one embodiment, the method of preparing the APCs further comprisesexpanding the immune cells in the presence of a growth factor. In oneembodiment, the method of preparing the APCs further comprises expandingthe activated APCs in the presence of a growth factor. In one aspect,the growth factor is a cytokine. In another aspect, the growth factorincludes, but is not limited to, Flt-3 ligand, GM-CSF, IL-4, M-CSF,IFNα, IL-1β, IL-4, IL-6, IL-13, IL-15, TNFα, or any combination thereof.

The APCs (e.g., dendritic cells) may be used to treat any cancers. Morepreferably, the cancer exhibits a chemorepellant effect. Therefore, themethod of preparing the APCs may further comprise contacting theisolated antigen-presenting dendritic cells with an effective amount ofan anti-chemorepellant agent. Anti-chemorepellant agents may include,without limitation, molecules that inhibit expression of CXCL12 or CXCR4or CXCR7 (e.g., antisense or siRNA molecules), molecules that bind toCXCL12 or CXCR4 or CXCR7 and inhibit their function (e.g., antibodies oraptamers), molecules that inhibit dimerization of CXCL12 or CXCR4 orCXCR7, and antagonists of CXCR4 or CXCR7. In one embodiment, theinhibitor of CXCL12 signaling is a CXCR4 antagonist. In one aspect, theanti-chemorepellant agents include but are not limited to AMD3100 or aderivative thereof, AMD11070 (also called AMD070), AMD12118, AMD11814,AMD13073, FAMD3465, C131, BKT140, CTCE-9908, KRH-2731, TC14012,KRH-3955, BMS-936564/MDX-1338, LY2510924, GSK812397, KRH-1636, T-20,T-22, T-140, TE-14011, T-14012, TN14003, TAK-779, AK602, SCH-351125,tannic acid, NSC 651016, thalidomide, GF 109230X, an antibody thatinterferes with dimerization of a chemorepellant chemokine, such asCXCL12, an antibody that interferes with dimerization of a receptor fora chemorepellant chemokine, such as CXCR4 or CXCR7, or a combinationthereof. In another aspect, the anti-chemorepellant agent is AMD3100.AMD3100 is described in U.S. Pat. No. 5,583,131, which is incorporatedby reference herein in its entirety. In one embodiment, theanti-chemorepellant agent is a CXCR7 antagonist. The CXCR7 antagonistcan be but is not limited to CCX771, CCX754, or an antibody thatinterferes with the dimerization of CXCR7. In certain embodiments, theanti-chemorepellant agent is not an antibody. In certain embodiments,the anti-chemorepellant agent is not a heparinoid. In certainembodiments, the anti-chemorepellant agent is not a peptide. In oneembodiment, the amount of anti-chemorepellant agent is sufficient tobind to at least a subset of receptors, e.g., CXCR4 and/or CXCR7, on thesurface of the T cells.

In one embodiment, the APCs are allogeneic, autologous, or derived froma cell line. In another embodiment, the APCs are collected from apatient having a tumor. In one aspect, either the APCs or the tumorcells are immobilized on a solid support. The solid support includes butis not limited to a surface of a column, a sepharose bead, a gel, amatrix, a magnetic bead, a plastic surface, or any combination thereof.

In another embodiment, the dendritic cells are differentiated or maturedfrom dendritic cell precursors. The dendritic cell precursors may beactivated, or not be activated. In another aspect, the dendritic cellprecursors are free or substantially free of neutrophils, macrophages,lymphocytes, or a combination thereof. In one aspect, the dendritic cellprecursors are differentiated in the presence of a growth factor. Thegrowth factor may be a cytokine. In another aspect, the growth factor isselected from a group consisting of Flt-3 ligand, GM-CSF, IL-4, M-CSF,IFNα, IL-1β, IL-4, IL-6, IL-13, IL-15 and TNFα.

Another aspect of the invention relates to a method for treating acancer, e.g., a tumor, in a patient, which comprises administering aneffective amount of ex vivo prepared APCs to the patient, wherein theAPCs are prepared by incubating immune cells with cancer cells and afusion protein, wherein the fusion protein comprises a target or antigenbinding component and a heat shock protein component. In particular, thetarget or antigen binding component of the fusion protein may bind tothe cancer cells, and the heat shock protein component may activate theAPCs. The target or antigen binding component may be any antibody orother molecule that recognizes a cancer cell of interest. In one aspect,the target or antigen binding component is a single chain antibody, avariable domain fragment, or a Fab portion of an antibody. In anotheraspect, the target or antigen binding component is specific for a cancerantigen (e.g., a tumor-specific antigen or a tumor-associated antigen).The cancer antigen may be any identifiable cell surface antigen that isexpressed by a cancer of interest. In one embodiment, the tumor antigenis mesothelin, alphafetoprotein, CEA, CA-125, MUC-1, Her2Neu, ETA,NY-ESO-1, VEGF, VEGFR1, VEGFR2, PSMA, prostate specific antigen,HPV17E7, mutant p53, surviving, ras, MAGE, gp100, tyrosinase, WT1, PR1,folate-binding protein, CA-19-9, FAP, G250, or A33. In one embodiment,the antibody is specific for mesothelin. Fusion proteins that recognizeand bind to mesothelin are described, for example, in U.S. Pat. No.7,943,133, which is incorporated herein by reference in its entirety.

In one embodiment, the non-limiting examples of the immune cellsinclude, but are not limited to, dendritic cells, B lymphocytes,mononuclear phagocytes, and/or a combination thereof. The mononuclearphagocytes may be monocytes or macrophages. In another aspect, theimmune cells bear at least a CD40 receptor. In another aspect, theimmune cells are dendritic cells.

The methods described herein may be used to treat the cancer. In oneembodiment, the cancer exhibits a chemorepellant effect. In oneembodiment, the chemorepellant effect is mediated by overexpression ofCXCL12 (e.g., at a concentration of 100 nM or greater) or otherchemorepellant chemokine.

In one embodiment, the cancer comprises breast cancer, head and neckcancer, leukocytic cancer, liver cancer, ovarian cancer, bladder cancer,prostatic cancer, skin cancer, bone cancer, brain cancer, leukemiacancer, lung cancer, colon cancer, anal cancer, CNS cancer, melanomacancer, renal cancer, cervical cancer, esophageal cancer, testicularcancer, spleenic cancer, kidney cancer, lymphatic cancer, pancreaticcancer, stomach cancer or thyroid cancer. In one embodiment, the canceris mesothelioma. In one embodiment, the cancer is a hematologicalmalignancy. Hematological malignancies include tumors of the blood, bonemarrow, lymph, and lymphatic system, including, but not limited to,leukemias, lymphomas, and myelomas. In one embodiment, the cancer is ahuman papilloma virus (HPV)-positive cancer.

In one embodiment, the method further comprises selecting a patienthaving a cancer that exhibits a chemorepellant effect. In anotherembodiment, the invention further comprises administering to the patientan effective amount of an anti-chemorepellant agent. Theanti-chemorepellant agent and the APCs, including dendritic cells, maybe administered separately, simultaneously, and/or sequentially. Inanother aspect, the anti-chemorepellant agent is administered after theadministration of the APCs, including dendritic cells. In anotheraspect, the anti-chemorepellant agent is administered before and/orduring the administration of the APCs (e.g., dendritic cells). In oneaspect, the anti-chemorepellant agent is administered via injection. Inone embodiment, the anti-chemorepellant agent is administered directlyor proximal to the tumor. In one embodiment, the anti-chemorepellantagent is administered systemically.

In one embodiment, the APCs have an anti-chemorepellant agent boundthereto via one or more cell surface receptors. The non-limitingexamples of the anti-chemorepellant agents include but are not limitedto AMD3100 or a derivative thereof, AMD11070 (also called AMD070),AMD12118, AMD11814, AMD13073, FAMD3465, C131, BKT140, CTCE-9908,KRH-2731, TC14012, KRH-3955, BMS-936564/MDX-1338, LY2510924, GSK812397,KRH-1636, T-20, T-22, T-140, TE-14011, T-14012, TN14003, TAK-779, AK602,SCH-351125, tannic acid, NSC 651016, thalidomide, GF 109230X, anantibody that interferes with dimerization of a chemorepellantchemokine, an antibody that interferes with dimerization of a receptorfor a chemorepellant chemokine, and/or a combination thereof. In oneaspect, the anti-chemorepellant agent is AMD3100.

In one embodiment, the heat shock protein component may be HSP70 or animmune activating fragment and/or modified sequence thereof. The HSP70or the immune activating fragment and/or modified sequence thereof maybe from Mycobacterium tuberculosis. In another aspect, the target orantigen binding component comprises a single chain antibody, a variabledomain, or a Fab domain.

In another embodiment, the method of treating cancer in a patientfurther comprises expanding the immune cells, e.g., dendritic cells, inthe presence of a growth factor. In one aspect, the growth factor is acytokine. In another aspect, the non-limiting examples of the growthfactors include Flt-3 ligand, GM-CSF, IL-4, M-CSF, IFNα, IL-1β, IL-4,IL-6, IL-13, IL-15, TNFα, and a combination thereof.

In one embodiment, the tumor cells are obtained from the patient. Inanother embodiment, the tumor cells derive from breast cancer, head andneck cancer, leukocytic cancer, liver cancer, ovarian cancer, bladdercancer, prostatic cancer, skin cancer, bone cancer, brain cancer,leukemia cancer, lung cancer, colon cancer, anal cancer, CNS cancer,melanoma cancer, renal cancer, cervical cancer, esophageal cancer,testicular cancer, spleenic cancer, kidney cancer, lymphatic cancer,pancreatic cancer, stomach cancer or thyroid cancer. In one embodiment,the tumor cells derive from mesothelioma. In one embodiment, the tumorcells are from a hematological malignancy. In one embodiment, the canceris a HPV-positive cancer.

In one embodiment, the APCs (e.g., dendritic cells) are allogeneic,autologous, or derived from a cell line. In one aspect, the APCs (e.g.,dendritic cells) are collected from a patient having a tumor.

In another embodiment, the immune cells, APCs (e.g., dendritic cells),or the tumor cells are immobilized on a solid support. In one aspect,the solid support comprises a column, a sepharose bead, a gel, a matrix,a magnetic bead, or a plastic surface.

In one embodiment, the dendritic cells are differentiated or maturedfrom dendritic cell precursors. In another aspect, the dendritic cellprecursors are non-activated or activated. In one aspect, the dendriticcell precursors are free or substantially free of neutrophils,macrophages, lymphocytes, or a combination thereof. In another aspect,the dendritic cell precursors are differentiated in the presence of agrowth factor. The growth factor may be a cytokine. In another aspect,the non-limiting examples of the growth factor include but are notlimited to Flt-3 ligand, GM-CSF, IL-4, M-CSF, IFNα, IL-1β, IL-4, IL-6,IL-13, IL-15, and TNFα.

In one aspect, this disclosure relates to a method for preparingactivated T cells, the method comprising:

a) providing activated antigen-presenting cells that were activated inpresence of cancer or tumor cells and a fusion protein ex vivo for atime period sufficient to produce activated antigen-presenting cells,wherein at least one activated antigen-presenting cell displays anantigen derived from the cancer or tumor cells;

b) contacting the activated antigen-presenting cells with T cells for aperiod of time sufficient to activate the T cells; and

c) isolating the activated T cells;

wherein the fusion protein comprises a cancer or tumor binding componentand a heat shock protein component, wherein said cancer or tumor bindingcomponent binds to the cancer tumor cells and said heat shock proteincomponent activates antigen-presenting cells. In one embodiment, theisolated activated T cells are free or substantially free of thedendritic cells, cancer or tumor cells and the fusion protein.

In one embodiment, the heat shock protein component comprises HSP70 oran immune activating fragment and/or modified sequence thereof. In oneembodiment, the HSP70 or the immune activating fragment and/or modifiedsequence thereof is from Mycobacterium tuberculosis. In one embodiment,the tumor binding component is a single chain antibody, a variabledomain, or a Fab domain.

In one embodiment, the APCs were expanded in the presence of a growthfactor. In one embodiment, the growth factor is a cytokine. In oneembodiment, the growth factor is selected from the group consisting ofFlt-3 ligand, GM-CSF, IL-4, M-CSF, IFNα, IL-1β, IL-4, IL-6, IL-13, IL-15and TNFα.

In one embodiment, the tumor cells are obtained from a patient. In oneembodiment, the tumor cells are derived from a patient to be treated asdescribed herein. In one embodiment, the tumor cells are derived from adifferent patient. In one embodiment, the tumor cells comprise a cancercell line.

In one embodiment, the term “tumor cells” that are incubated with theimmune cells and fusion protein refers to any tumor sample, includingbut not limited to intact cells, viable cells, non-viable cells,tumor-specific antigen (e.g., isolated antigen, partially isolatedantigen, recombinant antigen, etc.), and/or other cellular materialderived from a tumor or cancer cell or cell line. In some embodiments,the tumor sample comprises at least a subset of antigens that areidentical or similar to antigens associated with a patient's tumor,e.g., that will result in a T cell response against the patient's tumor.

Anti-chemorepellant agents may include, without limitation, moleculesthat inhibit expression of CXCL12 or CXCR4 or CXCR7 (e.g., antisense orsiRNA molecules), molecules that bind to CXCL12 or CXCR4 or CXCR7 andinhibit their function (e.g., antibodies or aptamers), molecules thatinhibit dimerization of CXCL12 or CXCR4 or CXCR7, and antagonists ofCXCR4 or CXCR7. In one embodiment, the inhibitor of CXCL12 signaling isa CXCR4 antagonist. In one embodiment, the isolated activated T cellsare contacted with an effective amount of an anti-chemorepellant agent.In one embodiment, the anti-chemorepellant agent is selected from thegroup consisting of AMD3100 or a derivative thereof, AMD11070 (alsocalled AMD070), AMD12118, AMD11814, AMD13073, FAMD3465, C131, BKT140,CTCE-9908, KRH-2731, TC14012, KRH-3955, BMS-936564/MDX-1338, LY2510924,GSK812397, KRH-1636, T-20, T-22, T-140, TE-14011, T-14012, TN14003,TAK-779, AK602, SCH-351125, tannic acid, NSC 651016, thalidomide, GF109230X, an antibody that interferes with dimerization of achemorepellant chemokine, such as CXCL12, and an antibody thatinterferes with dimerization of a receptor for a chemorepellantchemokine, such as CXCR4 or CXCR7. In one embodiment, theanti-chemorepellant agent is AMD3100. AMD3100 is described in U.S. Pat.No. 5,583,131, which is incorporated by reference herein in itsentirety. In one embodiment, the anti-chemorepellant agent is a CXCR7antagonist. The CXCR7 antagonist can be but is not limited to CCX771,CCX754, or an antibody that interferes with the dimerization of CXCR7.In certain embodiments, the anti-chemorepellant agent is not anantibody. In certain embodiments, the anti-chemorepellant agent is not aheparinoid. In certain embodiments, the anti-chemorepellant agent is nota peptide. In one embodiment, the amount of anti-chemorepellant agent issufficient to bind to at least a subset of receptors, e.g., CXCR4 and/orCXCR7, on the surface of the T cells.

In one embodiment, the APCs are allogeneic, autologous, or derived froma cell line. In one embodiment, the T cells are allogeneic, autologous,or derived from a cell line. In one embodiment, the APCs and/or the Tcells are isolated from a patient having cancer. In some embodiments,the APCs and T cells are derived from the same source, e.g., the samepatient.

In one embodiment, either the APCs or the tumor cells are immobilized ona solid support. In one embodiment, the activated APCs are isolated byisolating the solid support from the tumor cells and fusion protein.

In one embodiment, the T cells are immobilized on a solid support. Inone embodiment, the activated T cells are isolated by isolating thesolid support from the APCs.

In one embodiment, the solid support comprises a surface of a column, asepharose bead, a gel, a matrix, a magnetic bead, or a plastic surface.

In one embodiment, this disclosure relates to a method for treating acancer, e.g., a tumor, in a patient, the method comprising administeringan effective amount of activated T cells to the patient, wherein theactivated T cells were prepared by:

-   -   providing activated APCs prepared by incubating APCs with cancer        cells and a fusion protein, wherein the fusion protein comprises        a cancer cell binding component and a heat shock protein        component, wherein said cancer binding component binds to the        cancer cells and said heat shock protein component activates        APCs; and    -   contacting the activated APCs with T cells for a period of time        sufficient to activate the T cells.

In one embodiment, provided herein is a method for treating a cancer,e.g., a tumor, in a patient, the method comprising:

a) providing activated APCs that were activated by incubating APCs inthe presence of cancer cells and a fusion protein ex vivo for a timeperiod sufficient to produce activated APCs, wherein at least oneactivated APC displays an antigen derived from the cancer cells;

b) contacting the activated APCs with T cells for a period of timesufficient to activate the T cells;

c) isolating the activated T cells; and

d) administering an effective amount of the activated T cells to thepatient;

wherein the fusion protein comprises a cancer cell binding component anda heat shock protein component, wherein said cancer cell bindingcomponent binds to the cancer cells and said heat shock proteincomponent activates APCs. In one embodiment, the isolated activated Tcells are free or substantially free of the activated APCs, cancer cellsand the fusion protein.

In one embodiment, the method further comprises administering to thepatient an effective amount of an anti-chemorepellant agent. In oneembodiment, the activated T cells have an anti-chemorepellant agentbound thereto via one or more cell surface receptors. In one embodiment,the one or more cell surface receptors comprise CXCR4 and/or CXCR7.

In one embodiment, the anti-chemorepellant agent is one described above,e.g., AMD3100 or a derivative thereof, AMD11070 (also called AMD070),AMD12118, AMD11814, AMD13073, FAMD3465, C131, BKT140, CTCE-9908,KRH-2731, TC14012, KRH-3955, BMS-936564/MDX-1338, LY2510924, GSK812397,KRH-1636, T-20, T-22, T-140, TE-14011, T-14012, TN14003, TAK-779, AK602,SCH-351125, tannic acid, NSC 651016, thalidomide, GF 109230X, anantibody that interferes with dimerization of a chemorepellantchemokine, or an antibody that interferes with dimerization of areceptor for a chemorepellant chemokine. In one embodiment, theanti-chemorepellant agent is an AMD3100 derivative. In one embodiment,the anti-chemorepellant agent is AMD3100.

The methods described herein may be used to treat the cancer. In oneembodiment, the cancer exhibits a chemorepellant effect. In oneembodiment, the chemorepellant effect is mediated by overexpression ofCXCL12 or other chemorepellant chemokine.

In one embodiment, the cancer comprises breast cancer, leukocyticcancer, liver cancer, ovarian cancer, head and neck cancer, bladdercancer, prostatic cancer, skin cancer, bone cancer, brain cancer,leukemia cancer, lung cancer, colon cancer, anal cancer, CNS cancer,melanoma cancer, renal cancer, cervical cancer, esophageal cancer,testicular cancer, spleenic cancer, kidney cancer, lymphatic cancer,pancreatic cancer, stomach cancer or thyroid cancer. In one embodiment,the cancer is mesothelioma. In one embodiment, the cancer is ahematological malignancy. Hematological malignancies include cancers ofthe blood, bone marrow, lymph, and lymphatic system, including, but notlimited to, leukemias, lymphomas, and myelomas. In one embodiment, thecancer is a HPV-positive cancer.

In one embodiment, this invention relates to a composition comprisingimmune cells and an effective amount of a fusion protein, said fusionprotein comprising a cancer cell binding component and a stress proteincomponent.

In one embodiment, this invention relates to a pharmaceuticalcomposition comprising activated antigen presenting cells and ananti-chemorepellant agent.

In one embodiment, the term “tumor cells” that are incubated with theimmune cells and fusion protein refers to any tumor sample, includingbut not limited to intact cells, viable cells, non-viable cells,tumor-specific antigen (e.g., isolated antigen, partially isolatedantigen, recombinant antigen, etc.), and/or other cellular materialderived from a tumor or cancer cell or cell line. In some embodiments,the tumor sample comprises at least a subset of antigens that areidentical or similar to antigens associated with a patient's tumor,e.g., that will result in a T cell response against the patient's tumor.

In one embodiment, provided herein is a pharmaceutical compositioncomprising activated APCs and an anti-chemorepellant agent. In oneembodiment, the APCs were activated by a method as described herein.

In one embodiment, the activated APCs have an anti-chemorepellant agentbound thereto via one or more cell surface receptors. In one embodiment,the one or more cell surface receptors comprise CXCR4 and/or CXCR7.

In one embodiment, provided herein is a pharmaceutical compositioncomprising activated T cells and an anti-chemorepellant agent. In oneembodiment, the T cells were activated by a method as described herein.

In one embodiment, the activated T cells have an anti-chemorepellantagent bound thereto via one or more cell surface receptors. In oneembodiment, the one or more cell surface receptors comprise CXCR4 orCXCR7.

In one embodiment, the composition further comprises a pharmaceuticallyacceptable excipient. In one embodiment, the composition furthercomprises an effective amount of a fusion protein as described herein.

In one embodiment is provided a composition comprising a complexincluding an activated APC, a fusion protein, and a T cell. In oneembodiment, the fusion protein comprises a cancer cell binding componentand a stress protein component. In one embodiment, the T cell isactivated by the interaction with the activated APC and/or fusionprotein. In one embodiment, the APC comprises an antigen derived from acancer sample on the cell surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the peptide sequence of VIC-007 (SEQ ID NO.: 1) and VIC-008(SEQ ID NO.: 2).

FIG. 2A indicates intraperitoneal ovarian tumor growth usingbioluminescence signals for each treatment (saline, VIC-007, or VIC-008)at weeks 0 (W0), 1 (W1), 2 (W2), 3 (W3), 4 (W4) and 5 (W5).

FIG. 2B is a graphical representation of the tumor growth shown in FIG.2A.

FIG. 3 shows percent survival of the mice inoculated with ovarian cancercells, after administration of saline, VIC-007, or VIC-008.

FIG. 4 shows the treatment protocol for tumor inoculation (mesothelioma)followed by administration of saline, VIC-008 alone, AMD3100 alone, orVIC-008 with AMD3100.

FIG. 5 shows percent survival of the mice inoculated with mesotheliomacancer cells, Luc-40L, after administration of saline, VIC-008 alone,AMD3100 alone, or VIC-008 with AMD3100.

FIG. 6 shows percent survival of the mice inoculated with mesotheliomacancer cells, Luc-AE17, after administration of saline, VIC-008 alone,AMD3100 alone, or VIC-008 with AMD3100.

FIGS. 7A-7D show tumor growth and mice survival in different treatmentgroups. All the intraperitoneal tumors were collected at day 7 after thelast treatment and weighed in 40L (n=10) (A) and AE17 (n=10) (B) mice.The survival of 40L (n=12) (C) and AE17 (n=12) (D) tumor bearing mice.Numbers on X axis represented days of mouse survival after inoculationof tumor cells into mice. NS, not significant, *P<0.05,**P<0.01,***P<0.001 and ****P<0.0001. Data were presented as mean±SEM.

FIGS. 8A-8F show VIC-008 facilitates lymphocyte infiltration. (A) Gatingstrategy for infiltrated lymphocytes. The proportion of CD8⁺ T cells intotal live splenocytes in 40L (n=6) (B) and AE17 (n=5) (C) mice. Theproportion of CD8⁺ T cells in total live cells in lymph nodes of AE17mice (n=5) (D). The proportion of CD8⁺ T cells in total live cells intumors of 40L (n=6) (E) and AE17 (n=5) (F) mice. *P<0.05, **P<0.01 and***P<0.001. Data are presented as mean±SEM.

FIGS. 9A-9D show VIC-008 promoted CD8⁺ T-cell IFN-γ secretion. (A)Representative dot plots of IFN-γ secreting cells in different treatmentgroups. The proportion of IFN-γ secreting cells in CD8⁺ T cells inspleens of 40L (n=6) (B) and AE17 (n=5) (C) mice. The proportion ofIFN-γ secreting cells in CD8⁺ T cells in lymph nodes of AE17 mice (n=5)(D). **P<0.01 and ***P<0.001. Data are presented as mean±SEM.

FIGS. 10A-10E show AMD3100 decreased PD-1 expression on CD8⁺ T cells.The proportion of PD-1-expressing cells in CD8⁺ T cells in spleens of40L (n=6) (A) and AE17 (n=5) (B) mice. The proportion of PD-1-expressingcells in CD8⁺ T cells in lymph nodes of AE17 mice (n=5) (C). Theproportion of PD-1-expressing cells in CD8⁺ T cells in tumors of 40L(n=6) (D) and AE17 (n=5) (E) mice. *P<0.05 and **P<0.01. Data arepresented as mean±SEM.

FIGS. 11A-11F show AMD3100 reduced Tumor-infiltrating T_(reg). Theproportion of T_(reg) in total live splenocytes in 40L (n=6) (A) andAE17 (n=5) (B) mice. The proportion of T_(reg) in total live cells inlymph nodes of AE17 mice (n=5) (C). The ratio of CD8⁺ T cells to T_(reg)in lymph nodes of AE17 mice (n=5) (D). The proportion of T_(reg) intotal live cells in tumors of 40L mice (n=6) (E). The ratio of CD8⁺ Tcells to T_(reg) in tumors of 40L mice (n=6) (F). *P<0.05, **P<0.01,***P<0.001 and ****P<0.0001. Data are presented as mean ±SEM.

FIGS. 12A-12E show AMD3100 reprogrammed T_(reg) to helper-like cells.The ratio of CD25⁻ to CD25⁺ cells in CD4⁺ Foxp3⁺ T-cell population intumors of 40L (n=6) (A) and in lymph nodes of AE17 (n=5) (B) mice.Representative density plots of IL-2⁺ CD40L⁺ cells in Foxp3⁺ CD25⁻T_(reg) population in different treatment groups (C). The proportion ofIL-2⁺ CD40L⁺ cells in Foxp3⁺ CD25⁻ T_(reg) population in tumors of 40L(n=6) (D) and in lymph nodes of AE17 (n=5) (E) mice. *P<0.05,**P<0.01,***P<0.001 and ****P<0.0001. Data are presented as mean±SEM.

FIGS. 13A-13F show AMD3100-driven T_(reg) reprogramming required TCRactivation. (A) Representative dot plots of Foxp3⁺ CD25⁻ and Foxp3⁺CD25⁺ population with or without AMD3100 treatment under noanti-CD3/CD28 stimulation (A) and statistical difference was analyzedusing unpaired t-test with Welch's correction (n=4) (B). Representativedot plots of Foxp3⁺ CD25⁻ and Foxp3⁺ CD25⁺ population with or withoutAMD3100 treatment under anti-CD3/CD28 stimulation (C) and statisticaldifference was analyzed using unpaired t-test with Welch's correction(n=4) (D). Representative density plots of IL-2⁺ CD40L⁺ cells in Foxp3⁺CD25⁻ T_(reg) population with or without AMD3100 treatment underanti-CD3/CD28 stimulation (E) and statistical difference was analyzedusing unpaired t-test with Welch's correction (n=4) (F). Data arepresented as mean±SEM.

DETAILED DESCRIPTION

After reading this description, it will become apparent to one skilledin the art how to implement the invention in various alternativeembodiments and alternative applications. However, not all embodimentsof the present invention are described herein. It will be understoodthat the embodiments presented here are presented by way of an exampleonly, and not limitation. As such, this detailed description of variousalternative embodiments should not be construed to limit the scope orbreadth of the present invention as set forth below.

Before the present invention is disclosed and described, it is to beunderstood that the aspects described below are not limited to specificcompositions, methods of preparing such compositions, or uses thereof assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting the invention. Asused herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

All numerical designations, e.g., pH, temperature, time, concentration,amounts, and molecular weight, including ranges, are approximationswhich are varied (+) or (−) by 10%, 1%, or 0.1%, as appropriate. It isto be understood, although not always explicitly stated, that allnumerical designations may be preceded by the term “about.” It is alsoto be understood, although not always explicitly stated, that thereagents described herein are merely exemplary and that equivalents ofsuch are known in the art.

The term “antigen-presenting cell” or “APC” refers to an immune cellthat is capable of presenting an antigen to the immune system includingdendritic cells, mononuclear phagocytes (e.g., monocytes andmacrophages), B lymphocytes, and the like, from a mammal (e.g., human ormouse). The antigen may be presented on the surfaces of theantigen-presenting cells.

The term “dendritic cell” refers to one type of antigen-presenting cellcapable of activating T cells and/or stimulating the differentiation ofB cells.

The term “CD40 receptor” refers to a costimulatory protein, peptide, orpolypeptide, or a nucleic acid encoding the peptide, polypeptide orprotein. The CD40 receptor may be present in immune cells, including butnot limited to APCs.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “comprising” or “comprises” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of,” when used to define compositionsand methods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this invention.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In one embodiment, the patient, subject, or individual is a mammal. Insome embodiments, the mammal is a mouse, a rat, a guinea pig, anon-human primate, a dog, a cat, or a domesticated animal (e.g., horse,cow, pig, goat, sheep). In some embodiments, the patient, subject orindividual is a human.

The term “treating” or “treatment” covers the treatment of a disease ordisorder described herein, in a subject, such as a human, and includes:(i) inhibiting a disease or disorder, i.e., arresting its development;(ii) relieving a disease or disorder, i.e., causing regression of thedisease or disorder; (iii) slowing progression of the disease ordisorder; and/or (iv) inhibiting, relieving, or slowing progression ofone or more symptoms of the disease or disorder. For example, treatmentof a cancer or tumor includes, but is not limited to, reduction in sizeof the tumor, elimination of the tumor and/or metastases thereof,remission of the cancer, inhibition of metastasis of the tumor,reduction or elimination of at least one symptom of the cancer, and thelike.

The term “administering” or “administration” of an agent to a subjectincludes any route of introducing or delivering to a subject a compoundto perform its intended function. Administration can be carried out byany suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

“Pharmaceutically acceptable composition” refers to a composition thatis suitable for administration to a mammal, particularly, a human.

It is also to be appreciated that the various modes of treatment orprevention of medical diseases and conditions as described are intendedto mean “substantial,” which includes total but also less than totaltreatment or prevention, and wherein some biologically or medicallyrelevant result is achieved.

The phrase “concurrently administering” refers to administration of atleast two agents to a patient over a period of time. As used herein, theword “concurrently” means sufficiently close in time to produce acombined effect (that is, concurrently can be simultaneously, or it canbe two or more events occurring within a short time period before orafter each other). Concurrent administration includes, withoutlimitation, separate, sequential, and simultaneous administration.

The term “separate” administration refers to an administration of atleast two active ingredients at the same time or substantially the sametime by different routes or in different compositions.

The term “sequential” administration refers to administration of atleast two active ingredients at different times, the administrationroute being identical or different. More particularly, sequential userefers to the whole administration of one of the active ingredientsbefore administration of the other or others commences. It is thuspossible to administer one of the active ingredients over severalminutes, hours, or days before administering the other active ingredientor ingredients.

The term “simultaneous” administration refers to the administration ofat least two active ingredients by the same route and at the same timeor at substantially the same time.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by suppression, remission, or eradication of adisease state.

The term “prevent” or “preventative” as used herein means a prophylactictreatment. A preventative effect is obtained by delaying the onset of adisease state or decreasing the severity of a disease state when itoccurs.

The term “therapeutically effective amount,” “prophylactically effectiveamount,” or “effective amount” refers to an amount of the agent that,when administered, is sufficient to cause the desired effect. Forexample, an effective amount of an anti-chemorepellant agent may be anamount sufficient to have an anti-chemorepellant effect on a cancer cellor tumor (e.g., to attenuate a chemorepellant effect from the tumor orcancer cell). The therapeutically effective amount of the agent may varydepending on the tumor being treated and its severity as well as theage, weight, etc., of the patient to be treated. The skilled artisanwill be able to determine appropriate dosages depending on these andother factors. The compositions can also be administered in combinationwith one or more additional therapeutic compounds. In the methodsdescribed herein, the therapeutic compounds may be administered to asubject having one or more signs or symptoms of a disease or disorder.

The term “kill” with respect to a cell/cell population is directed toinclude any type of manipulation that will lead to the death of thatcell/cell population.

The terms “antibodies” and “antibody” as used herein include polyclonal,monoclonal, single chain, chimeric, humanized and human antibodies,prepared according to conventional methodology.

The term “CXCR4/CXCL12 antagonist” or “CXCR7/CXCL12 antagonist” refersto a compound that antagonizes CXCL12 binding to CXCR4 and/or CXCR7 orotherwise reduces the fugetactic effect of CXCL12.

By “chemorepellant activity” or “chemorepellant effect” it is meant theability of an agent to repel (or chemorepel) a eukaryotic cell withmigratory capacity (i.e., a cell that can move away from a repellantstimulus), as well as the chemorepellant effect of a chemokine secretedby a cell, e.g., a tumor cell. Usually, the chemorepellant effect ispresent in an area around the cell wherein the concentration of thechemokine is sufficient to provide the chemorepellant effect. Somechemokines, including interleukin 8 and CXCL12, may exert chemorepellantactivity at high concentrations (e.g., over about 100 nM), whereas lowerconcentrations exhibit no chemorepellant effect and may even bechemoattractant.

Accordingly, an agent with chemorepellant activity is a “chemorepellantagent.” Such activity can be detected using any of a variety of systemswell known in the art (see, e.g., U.S. Pat. No. 5,514,555 and U.S.Patent Application Pub. No. 2008/0300165, each of which is incorporatedby reference herein in its entirety). A preferred system for use hereinis described in U.S. Pat. No. 6,448,054, which is incorporated herein byreference in its entirety.

The term “anti-chemorepellant effect” refers to the effect of theanti-chemorepellant agent to attenuate or eliminate the chemorepellanteffect of the chemokine.

The term “autogeneic” or “autologous” refers to the origin of a cell,when the cell administered to an individual is derived from theindividual or a genetically identical individual (i.e., an identicaltwin of the individual). An autogeneic cell can also be a progeny of anautogeneic cell. The term also indicates that cells of different celltypes are derived from the same individuals or genetically identicalindividuals.

The term “allogeneic” or “allogenic” also refers to the origin of acell, when the cell being administered to an individual is derived froman individual not genetically identical to the recipient but from thesame species, including a progeny of an allogeneic cell. Cells are stillcalled allogeneic when they are of different cell types but are derivedfrom genetically non-identical donors, or when they are progenies ofcells derived from genetically non-identical donors.

“Immune cells” as used herein are cells of hematopoietic origin that areinvolved in the specific recognition of antigens. Immune cells includeAPCs, such as dendritic cells or macrophages, B cells, T cells, etc.

The term “anti-cancer therapy” as used herein refers to traditionalcancer treatments, including chemotherapy and radiotherapy, as well asimmunotherapy and vaccine therapy.

The term “engineered antibody” refers to a recombinant molecule thatcomprises at least an antibody fragment comprising an antigen bindingsite derived from the variable domain of the heavy chain and/or lightchain of an antibody and may optionally comprise the entire or part ofthe variable and/or constant domains of an antibody from any of the Igclasses (e.g., IgA, IgD, IgE, IgG, IgM and IgY).

The term “epitope” refers to the region of an antigen to which anantibody binds preferentially and specifically. A monoclonal antibodybinds preferentially to a single specific epitope of a molecule that canbe molecularly defined. In the present invention, multiple epitopes canbe recognized by a multispecific antibody.

A “fusion protein” or “fusion polypeptide” refers to a hybridpolypeptide which comprises polypeptide portions from at least twodifferent polypeptides. The portions may be from proteins of the sameorganism, in which case the fusion protein is said to be “intraspecies,”“intragenic,” etc. In various embodiments, the fusion polypeptide maycomprise one or more amino acid sequences linked to a first polypeptide.In the case where more than one amino acid sequence is fused to a firstpolypeptide, the fusion sequences may be multiple copies of the samesequence, or alternatively, may be different amino acid sequences. Afirst polypeptide may be fused to the N-terminus, the C-terminus, or theN- and C-terminus of a second polypeptide. Furthermore, a firstpolypeptide may be inserted within the sequence of a second polypeptide.

The term “immunogenic” refers to the ability of a substance to elicit animmune response. An “immunogenic composition” or “immunogenic substance”is a composition or substance which elicits an immune response. An“immune response” refers to the reaction of a subject to the presence ofan antigen, which may include at least one of the following: makingantibodies, developing immunity, developing hypersensitivity to theantigen, and developing tolerance.

The term “linker” is art-recognized and refers to a molecule or group ofmolecules connecting two compounds, such as two polypeptides. The linkermay be comprised of a single linking molecule or may comprise a linkingmolecule and a spacer molecule, intended to separate the linkingmolecule and a compound by a specific distance.

As used herein, a “stress protein” also known as a “heat shock protein”or “HSP” is a protein that is encoded by a stress gene, and is thereforetypically produced in significantly greater amounts upon the contact orexposure of the stressor to the organism. The term “stress protein” asused herein is intended to include such portions and peptides of astress protein. A “stress gene,” also known as “heat shock gene,” asused herein, refers to a gene that is activated or otherwise detectablyupregulated due to the contact or exposure of an organism (containingthe gene) to a stressor, such as but not limited to heat shock, hypoxia,glucose deprivation, heavy metal salts, inhibitors of energy metabolismand electron transport, and protein denaturants, or to certainbenzoquinone ansamycins. Nover, L., Heat Shock Response, CRC Press,Inc., Boca Raton, Fla. (1991). “Stress gene” also includes homologousgenes within known stress gene families, such as certain genes withinthe HSP70 and HSP90 stress gene families, even though such homologousgenes are not themselves induced by a stressor. Each of the terms stressgene and stress protein as used in the present specification may beinclusive of the other, unless the context indicates otherwise.

The term “vaccine” refers to a substance that elicits an immune responseand also confers protective immunity upon a subject.

Activated Antigen-Presenting Cells (APCs)

Antigen-Presenting Cells (APCs)

The antigen-presenting cells (APCs) may include dendritic cells, Blymphocytes, mononuclear phagocytes (e.g., monocytes and/ormacrophages), and/or other cell types expressing the necessaryMHC/co-stimulatory molecules. In one aspect, the APCs comprise acostimulatory protein, e.g., CD40, which is important for the activationof the APCs. In one embodiment, the APCs may be presented with antigensby pulsing, in which the APCs are exposed to antigenic proteins orpolypeptides. The proteins or peptides may be recombinant or naturalproducts. In another embodiment, APCs are exposed to the nucleic acidsencoding the proteins or peptides in the presence of transfection agentsknown in the art, including but not limited to cationic lipids.

Transfected or pulsed APCs can subsequently be administered to the hostvia an intravenous, subcutaneous, intranasal, intramuscular, orintraperitoneal route of delivery. Protein/peptide antigens can also bedelivered in vivo with adjuvant via the intravenous, subcutaneous,intranasal, intramuscular or intraperitoneal route of delivery.

According to the invention, foster antigen-presenting cells may also beused as a substitute for the antigen-presenting cells. The fosterantigen-presenting cells lack antigen processing activity, whereby theyexpress MHC molecules free of bound peptides. In one aspect, foster APCsare derived from the human cell line 174X CEM.T2, referred to as T2,which contains a mutation in its antigen processing pathway thatrestricts the association of endogenous peptides with cell surface MHCclass I molecules. Zweerink et al., J. Immunol. 150:1763-1771(1993).Transduction of T2 cells with specific recombinant MHC alleles allowsfor redirection of the MHC restriction profile. Libraries tailored tothe recombinant allele will be preferentially presented by them becausethe anchor residues will prevent efficient binding to the endogenousallele. High level expression of MHC molecules makes the APCs morevisible to the CTLs. Expressing the MHC allele of interest in T2 cellsusing a powerful transcriptional promoter (e.g., the CMV promoter)results in a more reactive APC.

In one embodiment, the APCs are modified to express or over-expressco-stimulatory molecules. Co-stimulatory molecules include, but are notlimited to, CD40, MHC class I and/or class II, B7-1 (CD80), B7-2 (CD86),ICAM-1, interleukin 1 (IL-1), and LFA-3. See, e.g., Hodge et al. J NatlCancer Inst (2000) 92 (15): 1228-1239, which is incorporated herein byreference in its entirety.

Dendritic Cells

As one type of antigen-presenting cells, dendritic cells are effectivein presenting antigens and initiating immune responses, particularly theimmune responses associated with T cells. Dendritic cells are present inskin, nose, lung, intestines, blood, and many other tissues and organs.The immunogenicity of the dendritic cells is partly due to their abilityto process antigen materials and present them on the cell surface.

After capturing the antigens, the dendritic cells can present theantigen to T cells. The dendritic cells become mature with increasedsurface expression of class II MHC and costimulatory molecules.Santambrogio et al., PNAS, 96(26): 15056-15061 (1999). Mature dendriticcells may lose the ability to further process exogenous antigens.Maturation of the dendritic cells may also be accompanied with the lossof acidic organelles, where the antigens were processed. Kampgen et al.,PNAS, 88: 3014-3018 (1991).

The efficiency of the dendritic cells may be limited by the small numberof the cells in any given organs. For example, around 0.1% of the whitecells exist as dendritic cells in human blood. Dendritic cells from someorgans (e.g., spleen and afferent lymphatic) arise from precursors.Thus, in one aspect of the invention, dendritic cells are obtained orexpanded from dendritic cell precursors. The methods of expanding orobtaining dendritic cells are described in U.S. Pat. No. 5,994,126,which is incorporated herein by reference in its entirety.

Methods of Making Activated Antigen-Presenting Cells

The activated APCs used in the methods described herein to activate Tcells are activated using a fusion protein and tumor cells or a tumorsample.

In one embodiment, the APCs used in the methods described herein areactivated by incubating an effective amount of a fusion protein withimmune cells and tumor cells or sample under conditions so as to produceactivated APCs. In one embodiment, the APCs are activated by a methodwhich comprises incubating immune cells ex vivo in the presence of tumorcells and a fusion protein for a time period sufficient to produce theactivated APCs, wherein at least one activated APC displays an antigenderived from the tumor cells. In one embodiment, the method furthercomprises isolating the activated APCs. In one embodiment, the isolatedactivated APCs are free or substantially free of the tumor cells and/orthe fusion protein.

The term “substantially free” as used herein refers to nearly completeremoval of the tumor cells and/or fusion protein. In one embodiment, theactivated APCs are free of the tumor cells and/or fusion protein. In oneembodiment, the activated APCs are free of the tumor cells. In oneembodiment, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or at least about 99.9% of the tumor cells are removed from theactivated APCs by isolation. In one embodiment, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or at least about 99.9% of thefusion protein is removed from the activated APCs by isolation.

In one aspect, the fusion protein comprises an antigen-binding componentand a heat shock protein component. While the antigen-binding componentbinds to the tumor cell, the heat shock protein component activates theAPCs. In one embodiment, the antigen-binding component of the fusionprotein may be any antibody or other molecule that recognizes a tumor orcancer cell of interest. In one embodiment, the antigen-bindingcomponent is a single chain antibody, a variable domain, or a fragmentantigen-binding (“Fab”) domain of an antibody.

In one aspect, the tumor cells are obtained from a patient.

In one aspect, the antigen-binding component is specific for a cancerantigen (e.g., a tumor-specific antigen or a tumor-associated antigen).The cancer antigen may be any identifiable cell surface antigen that isexpressed by a cancer of interest. In one embodiment, the cancer antigenis mesothelin, alphafetoprotein, CEA, CA-125, MUC-1, Her2Neu, ETA,NY-ESO-1, VEGF, VEGFR1, VEGFR2, PSMA, prostate specific antigen,HPV17E7, mutant p53, surviving, ras, MAGE, gp100, tyrosinase, WT1, PR1,folate-binding protein, CA-19-9, FAP, G250, or A33. In one embodiment,the antibody is specific for mesothelin. Fusion proteins that recognizeand bind to mesothelin are described, for example, in U.S. Pat. No.7,943,133, which is incorporated herein by reference in its entirety.

In one embodiment, the immune cells (APCs) include, but are not limitedto, dendritic cells, B lymphocytes, and mononuclear phagocytes. Themononuclear phagocytes may include monocytes and/or macrophages. In someaspects, the immune cells comprise at least one CD40. In another aspect,the immune cells are dendritic cells.

In one embodiment, the heat shock protein component includes but is notlimited to HSP70 and/or an immune activating fragment and/or modifiedsequence thereof. In another aspect, the HSP70 or the immune activatingfragment and/or modified sequence thereof is from Mycobacteriumtuberculosis.

In one embodiment, the method of preparing the APCs further comprisesexpanding the immune cells in the presence of a growth factor. In oneembodiment, the method of preparing the APCs further comprises expandingthe activated APCs in the presence of a growth factor. In one aspect,the growth factor is a cytokine. In another aspect, the growth factorincludes, but is not limited to, Flt-3 ligand, GM-CSF, IL-4, M-CSF,IFNα, IL-1β, IL-4, IL-6, IL-13, IL-15, TNFα, or any combination thereof.

In one embodiment, the APCs are allogeneic, autologous, or derived froma cell line. In another embodiment, the APCs are collected from apatient having a tumor. In one aspect, either the APCs or the tumorcells are immobilized on a solid support. The solid support includes butis not limited to a surface of a column, a sepharose bead, a gel, amatrix, a magnetic bead, a plastic surface, or any combination thereof.

In another embodiment, the dendritic cells are differentiated or maturedfrom dendritic cell precursors. The dendritic cell precursors may beactivated, or not be activated. In another aspect, the dendritic cellprecursors are free or substantially free of neutrophils, macrophages,lymphocytes, or a combination thereof. In such an aspect, the term“substantially free” indicates that neutrophils, macrophages, and/orlymphocytes comprise less than about 10%, e.g., less than about 5%,e.g., less than about 2%, e.g., less than about 1% of the total cells ina dendritic cell precursor composition.

In one aspect, the dendritic cell precursors are differentiated in thepresence of a growth factor. The growth factor may be a cytokine. Inanother aspect, the growth factor is selected from a group consisting ofFlt-3 ligand, GM-CSF, IL-4, M-CSF, IFNα, IL-1β, IL-4, IL-6, IL-13, IL-15and TNFα.

In one embodiment, the APCs are modified to express or over-expressco-stimulatory molecules. Co-stimulatory molecules include, but are notlimited to, CD40, MHC class I and/or class II, B7-1 (CD80), B7-2 (CD86),ICAM-1, interleukin 1 (IL-1), and LFA-3. See, e.g., Hodge et al. J NatlCancer Inst (2000) 92 (15): 1228-1239, which is incorporated herein byreference in its entirety.

Anti-Chemorepellant Agents

Many tumors have chemorepellant effects, e.g., on immune cells, due tochemokines secreted by the tumor cells. High concentrations of thechemokines secreted by the tumor cells can have chemorepellant effectson cells, whereas lower concentrations do not have such effects or evenresult in chemoattraction. For example, T cells are repelled by CXCL12(SDF-1) by a concentration-dependent and CXCR4 receptor-mediatedmechanism.

The anti-chemorepellant agent may be any such agent known in the art,for example an anti-chemorepellant agent as described in U.S. PatentApplication Publication No. 2008/0300165, which is hereby incorporatedby reference in its entirety.

Anti-chemorepellant agents include any agents that specifically inhibitchemokine and/or chemokine receptor dimerization, thereby blocking thechemorepellant response to a chemorepellant agent. Certain chemokines,including IL-8 and CXCL12 can also serve as chemorepellants at highconcentrations (e.g., above 100 nM) where many of the chemokines existas a dimer. Dimerization of the chemokines elicits a differentialresponse in cells, causing dimerization of chemokine receptors, anactivity which is interpreted as a chemorepellant signal. Blocking thechemorepellant effect of high concentrations of chemokines secreted by atumor can be accomplished, for example, by anti-chemorepellant agentswhich inhibit chemokine dimer formation or chemokine receptor dimerformation. For example, antibodies that target and block chemokinereceptor dimerization, e.g., by interfering with the dimerizationdomains or ligand binding, can be anti-chemorepellant agents.Anti-chemorepellant agents that act via other mechanisms of action,e.g., that reduce the amount of chemorepellant cytokine secreted by thecells, inhibit dimerization, and/or inhibit binding of the chemokine toa target receptor, are also encompassed by the present invention. Wheredesired, this effect can be achieved without inhibiting the chemotacticaction of monomeric chemokine.

In other embodiments, the anti-chemorepellant agent is a CXCR4antagonist, CXCR7 antagonist, CXCR3 antagonist, CXCR4/CXCL12 antagonist,CXCR7/CXCL12 antagonist, or selective PKC inhibitor. Anti-chemorepellantagents may include, without limitation, molecules that inhibitexpression of CXCL12 or CXCR4 or CXCR7 (e.g., antisense or siRNAmolecules), molecules that bind to CXCL12 or CXCR4 or CXCR7 and inhibittheir function (e.g., antibodies or aptamers), molecules that inhibitdimerization of CXCL12 or CXCR4 or CXCR7, and antagonists of CXCR4 orCXCR7.

The CXCR4 antagonist can be but is not limited to AMD3100 (plerixafor)or a derivative thereof, AMD11070 (also called AMD070), AMD12118,AMD11814, AMD13073, FAMD3465, C131, BKT140, CTCE-9908, KRH-2731,TC14012, KRH-3955, BMS-936564/MDX-1338, LY2510924, GSK812397, KRH-1636,T-20, T-22, T-140, TE-14011, T-14012, or TN14003, derivatives thereof,or an antibody that interferes with the dimerization of CXCR4.Additional CXCR4 antagonists are described, for example, in U.S. PatentPub. No. 2014/0219952 and Debnath et al. Theranostics, 2013; 3(1):47-75, each of which is incorporated herein by reference in itsentirety, and include TG-0054 (burixafor), AMD3465, NIBR1816, AMD070,and derivatives thereof.

The CXCR3 antagonist can be but is not limited to TAK-779, AK602, orSCH-351125, or an antibody that interferes with the dimerization ofCXCR3.

The CXCR4/CXCL12 antagonist can be but is not limited to tannic acid,NSC 651016, or an antibody that interferes with the dimerization ofCXCR4 and/or CXCL12.

The CXCR7/CXCL12 antagonist can be but is not limited to CCX771, CCX754,or an antibody that interferes with the dimerization of CXCR7 and/orCXCL12.

The selective PKC inhibitor can be but is not limited to thalidomide orGF 109230X.

In one embodiment, the anti-chemorepellant agent is AMD3100(plerixafor). AMD3100 is described in U.S. Pat. No. 5,583,131, which isincorporated by reference herein in its entirety.

In one embodiment, the anti-chemorepellant agent is an AMD3100derivative. AMD3100 derivatives include, but are not limited to, thosefound in U.S. Pat. Nos. 7,935,692 and 5,583,131 (USRE42152), each ofwhich is incorporated herein by reference in its entirety.

In certain embodiments, the anti-chemorepellant agent is not anantibody. In certain embodiments, the anti-chemorepellant agent is not aheparinoid. In certain embodiments, the anti-chemorepellant agent is nota peptide.

In one embodiment, the anti-chemorepellant agent is coupled with amolecule that allows targeting of a tumor. In one embodiment, theanti-chemorepellant agent is coupled with (e.g., bound to) an antibodyspecific for the tumor to be targeted. In one embodiment, theanti-chemorepellant agent coupled to the molecule that allows targetingof the tumor is administered systemically.

In one embodiment, the anti-chemorepellant agent is administered incombination with an additional compound that enhances theanti-chemorepellant activity of the agent. In one embodiment, theadditional compound is granulocyte colony stimulating factor (“G-CSF”).In one embodiment, G-CSF is not administered.

Fusion Protein

This disclosure relates to fusion proteins comprising a stress proteincomponent and a target or antigen binding component, and methods ofusing the same. In particular, this disclosure relates to treating apatient having a disease, e.g., a cancer or a disease caused by apathogen, that can be recognized by a fusion protein as describedherein. Preferably, the disease expresses a chemorepellant activity suchthat immune cells are inhibited in the vicinity of the diseased cells,pathogen, or tumor cells.

Examples and methods of making fusion proteins contemplated in thepresent invention are described in U.S. Pat. Nos. 8,143,387 and7,943,133 and PCT Application Number PCT/US2017/021911, each of which isincorporated herein by reference in its entirety.

Stress Protein Component

The stress protein component (also referred to as the stress proteindomain) may comprise any polypeptide sequence that activates APCs. Insome embodiments, the polypeptide sequence is derived from a stressprotein. However, any APC-activating polypeptide is contemplated.

Any suitable stress protein (e.g., heat shock protein) can be used inthe fusion polypeptides of the present invention. For example, HSP60and/or HSP70 can be used. Turning to stress proteins generally, cellsrespond to a stressor (typically heat shock treatment) by increasing theexpression of a group of genes commonly referred to as stress, or heatshock, genes. Heat shock treatment involves exposure of cells ororganisms to temperatures that are one to several degrees Celsius abovethe temperature to which the cells are adapted. In coordination with theinduction of such genes, the levels of corresponding stress proteinsincrease in stressed cells.

In bacteria, the predominant stress proteins are proteins with molecularsizes of about 70 kDa and 60 kDa, which are commonly referred to asHSP70 and HSP60, respectively. Stress proteins appear to participate inimportant cellular processes such as protein synthesis, intracellulartrafficking, and assembly and disassembly of protein complexes. Itappears that the increased amounts of stress proteins synthesized duringstress serve primarily to minimize the consequences of induced proteinunfolding. Indeed, the pre-exposure of cells to mildly stressfulconditions that induce the synthesis of stress proteins affordsprotection to the cells from the deleterious effects of a subsequent,more extreme stress.

The major stress proteins appear to be expressed in every organism andtissue type examined so far. Also, it appears that stress proteinsrepresent the most highly conserved group of proteins identified todate. For example, when stress proteins in widely diverse organisms arecompared, HSP90 and HSP70 exhibit 50% or higher identity at the aminoacid level and share many similarities at non-identical positions.Similar or higher levels of homology exist between different members ofa particular stress protein family within species.

The stress proteins, particularly HSP 70, HSP 60, HSP 20-30 and HSP 10,are among the major determinants recognized by the host immune system inthe immune response to infection by Mycobacterium tuberculosis andMycobacterium leprae. However, individuals, including healthyindividuals with no history of mycobacterial infection or autoimmunedisease, also carry T cells that recognize both bacterial and human HSP60 epitopes. This system recognizing stress protein epitopes presumablyconstitutes an “early defense system” against invading organisms. Thesystem may be maintained by frequent stimulation by bacteria andviruses.

Families of stress genes and proteins for use in the fusion polypeptidesare those well known in the art and include, for example, HSP 100-200,HSP 100, HSP 90, Lon, HSP 70, HSP 60, TF55, HSP 40, FKBPs, cyclophilins,HSP 20-30, ClpP, GrpE, HSP 10, ubiquitin, calnexin, and proteindisulfide isomerases. In certain embodiments, the stress protein is HSP70 or HSP 60. In certain embodiments, the stress protein is a fragmentof Hsp70 or Hsp60 and/or a modified sequence of Hsp70 or Hsp60. As useherein, a “modified sequence” of a stress protein such as Hsp70 is asequence comprising one or more additions, deletion, or substitutionsthat retains at least 50% of at least one of the biological activity ofthe stress protein, e.g., the ability to stimulate antigen presentingcells, e.g., at least 50%, 60%, 70%, 80%, or more of the biologicalactivity. In some embodiments, the modified sequence has an enhancedbiological activity compared to the wild-type sequence. In someembodiments, the modified sequence is one disclosed in PCT ApplicationNo. PCT/US2017/021911, incorporated by reference herein in its entirety.

The examples of HSP 100-200 include Grp170 (for glucose-regulatedprotein). HSP 100 examples include mammalian HSP 110, yeast HSP 104,ClpA, C1pB, C1pC, C1pX and C1pY. HSP 90 examples include HtpG in E.coli, HSP 83 and Hsc83 in yeast, and HSP 90alpha, HSP 90beta and Grp94in humans. HSP 70 examples include HSP 72 and Hsc73 from mammaliancells, DnaK from bacteria, particularly mycobacteria such asMycobacterium leprae, Mycobacterium tuberculosis, and Mycobacteriumbovis (such as Bacille-Calmette Guerin, referred to herein as Hsp71),DnaK from Escherichia coli, yeast, and other prokaryotes, and BiP andGrp78. HSP 60 examples include HSP 65 from mycobacteria. Bacterial HSP60 is also commonly known as GroEL, such as the GroEL from E. coli. TF55examples include Tep1, TRiC and thermosome. HSP 40 examples include DnaJfrom prokaryotes such as E. coli and mycobacteria and HSJ1, HDJ1 and HSP40. FKBPs examples include FKBP12, FKBP13, FKBP25, and FKBP59, Fpr1 andNep1. Cyclophilin examples include cyclophilinsA, Band C. HSP 10examples include GroES and Cpn10.

In particular embodiments, the stress proteins of the present inventionare obtained from enterobacteria, mycobacteria (particularly M. leprae,M. tuberculosis, M. vaccae, M. smegmatis and M. bovis), E. coli, yeast,Drosophila, vertebrates, avians, chickens, mammals, rats, mice,primates, or humans.

In one embodiment, the stress protein comprises Mycobacteriumtuberculosis-derived heat shock protein 70 (MtbHsp70). MtbHsp70 is wellcharacterized and functions as a potent immune-activating adjuvant. Itstimulates monocytes and dendritic cells (DCs) to produce CC-chemokines,which attract antigen processing and presenting macrophages, DCs, andeffector T and B cells.

A fusion polypeptide may comprise an amino acid sequence that is atleast about 80%, 85%, 90%, 95%, 98%, or 99% identical to a stressprotein described herein.

A fusion polypeptide may comprise an amino acid sequence which is afragment and/or modification of the stress protein as described herein.

Target Binding Component

The target binding component of the fusion protein may be any moleculethat specifically binds an antigen associated with the disease to betreated. In certain embodiments, the target binding component is anantibody or a fragment thereof. The terms “antigen-binding,” “targetbinding,” and “tumor-binding” are used interchangeably herein.

In one aspect, the target binding component is a single chain antibody.In one aspect, the target binding component is a variable domainfragment. In one aspect, the target binding component is a Fab portionof an antibody.

In one aspect, the target binding component is specific for a cancerantigen (e.g., a tumor-specific antigen or a tumor-associated antigen),and may be referred to as an antigen binding component. The cancerantigen may be any identifiable cell surface antigen that is expressedby a cancer of interest. In one embodiment, the cancer antigen ismesothelin, alphafetoprotein, CEA, CA-125, MUC-1, Her2Neu, ETA,NY-ESO-1, VEGF, VEGFR1, VEGFR2, PSMA, prostate specific antigen,HPV17E7, mutant p53, surviving, ras, MAGE, gp100, tyrosinase, WT1, PR1,folate-binding protein, CA-19-9, FAP, G250, or A33. In one embodiment,the antibody is specific for mesothelin. Fusion proteins that recognizeand bind to mesothelin are described, for example, in U.S. Pat. No.7,943,133, which is incorporated herein by reference in its entirety.

Fusion Proteins Targeting Mesothelin

In one embodiment, the target binding component is specific formesothelin. MSLN is highly overexpressed on the surface of commonepithelial cancers, including epithelial malignant mesothelioma andovarian cancer, while expressed at relatively low levels only inmesothelial cells lining the pleura, pericardium, and peritoneum inhealthy individuals.

In theory, fusion of anti-MSLN scFv and MtbHsp70 takes advantage of theimmune-activating action of MtbHsp70 and the tumor-targeting activity ofthe scFv, which will yield anti-tumor responses against the broadestprofile of tumor antigens.

In one embodiment, the fusion protein comprises a peptide having atleast about 85% sequence homology to the peptide sequence of SEQ ID NO.:1 or SEQ ID NO.: 2. In one embodiment, the fusion protein comprises apeptide having at least about 90% sequence homology to the peptidesequence of SEQ ID NO.: 1 or SEQ ID NO.: 2. In one embodiment, thefusion protein comprises a peptide having at least about 91% sequencehomology to the peptide sequence of SEQ ID NO.: 1 or SEQ ID NO.: 2. Inone embodiment, the fusion protein comprises a peptide having at leastabout 92% sequence homology to the peptide sequence of SEQ ID NO.: 1 orSEQ ID NO.: 2. In one embodiment, the fusion protein comprises a peptidehaving at least about 93% sequence homology to the peptide sequence ofSEQ ID NO.: 1 or SEQ ID NO.: 2. In one embodiment, the fusion proteincomprises a peptide having at least about 94% sequence homology to thepeptide sequence of SEQ ID NO.: 1 or SEQ ID NO.: 2. In one embodiment,the fusion protein comprises a peptide having at least about 95%sequence homology to the peptide sequence of SEQ ID NO.: 1 or SEQ IDNO.: 2. In one embodiment, the fusion protein comprises a peptide havingat least about 96% sequence homology to the peptide sequence of SEQ IDNO.: 1 or SEQ ID NO.: 2. In one embodiment, the fusion protein comprisesa peptide having at least about 97% sequence homology to the peptidesequence of SEQ ID NO.: 1 or SEQ ID NO.: 2. In one embodiment, thefusion protein comprises a peptide having at least about 98% sequencehomology to the peptide sequence of SEQ ID NO.: 1 or SEQ ID NO.: 2. Inone embodiment, the fusion protein comprises a peptide having at leastabout 99% sequence homology to the peptide sequence of SEQ ID NO.: 1 orSEQ ID NO.: 2. In one embodiment, the fusion protein comprises a peptidehaving a sequence disclosed in PCT Application No. PCT/US2017/021911,incorporated by reference herein in its entirety, or a sequence havingat least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence homology to a sequence disclosed therein.

In one embodiment, the fusion protein retains the ability to bind acancer antigen. In one embodiment, the fusion protein retains theability to activate APCs. In one embodiment, the fusion protein retainsthe ability to facilitate production of activated APCs havingcancer-specific antigens on the APC cell surface.

In one embodiment, the fusion protein comprises the peptide sequence ofSEQ ID NO.: 1. In one embodiment, the fusion protein comprises thepeptide sequence of SEQ ID NO.: 2.

Activated T Cells

In one aspect, this disclosure relates to methods of preparingactivated, cancer-targeting T cells from APCs that were activated usinga fusion protein as described herein.

In one embodiment, this invention relates to methods of preparingactivated T cells, which comprises incubating T cells with activatedantigen-presenting cells (APCs) for a period of time sufficient toproduce activated T cells, wherein the APCs were prepared by incubatingimmune cells ex vivo in the presence of tumor cells and a fusion proteinfor a time period sufficient to produce the activated APCs, wherein atleast one activated APC displays an antigen derived from the tumorcells. In one embodiment, the method further comprises isolating theactivated T cells, wherein the isolated activated T cells are free orsubstantially free of the activated APCs, tumor cells and/or the fusionprotein. In one embodiment, the APCs comprise dendritic cells.

The term “substantially free” as used with respect to the activated Tcells refers to nearly complete removal of the APCs and/or tumor cellsand/or fusion protein. In one embodiment, the activated T cells are freeof the APCs and/or tumor cells and/or fusion protein. In one embodiment,the activated T cells are free of the tumor cells. In one embodiment, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or at leastabout 99.9% of the tumor cells are removed from the activated T cells byisolation. In one embodiment, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99%, or at least about 99.9% of the fusion protein isremoved from the activated T cells by isolation. In one embodiment, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or at leastabout 99.9% of the APCs are removed from the activated T cells byisolation.

In one aspect, the fusion protein comprises an antigen-binding componentand a heat shock protein component. While the antigen-binding componentbinds to the tumor cells, the heat shock protein component activates theAPCs. In one embodiment, the antigen-binding component of the fusionprotein may be any antibody or other molecule that recognizes a tumor orcancer cell of interest. In one embodiment, the antigen-bindingcomponent is a single chain antibody, a variable domain, or a fragmentantigen-binding (“Fab”) domain of an antibody.

In one aspect, the tumor cells are obtained from a patient.

In one aspect, the antigen-binding binding component is specific for acancer antigen (e.g., a tumor-specific antigen or a tumor-associatedantigen). The cancer antigen may be any identifiable cell surfaceantigen that is expressed by a cancer of interest. In one embodiment,the cancer antigen is mesothelin, alphafetoprotein, CEA, CA-125, MUC-1,Her2Neu, ETA, NY-ESO-1, VEGF, VEGFR1, VEGFR2, PSMA, prostate specificantigen, HPV17E7, mutant p53, surviving, ras, MAGE, gp100, tyrosinase,WT1, PR1, folate-binding protein, CA-19-9, FAP, G250, or A33. In oneembodiment, the antibody is specific for mesothelin. Fusion proteinsthat recognize and bind to mesothelin are described, for example, inU.S. Pat. No. 7,943,133, which is incorporated herein by reference inits entirety.

In one embodiment, the antigen-presenting cells include, but are notlimited to, dendritic cells, B lymphocytes, and mononuclear phagocytes.The mononuclear phagocytes may include monocytes and/or macrophages. Insome aspects, the APCs comprise at least one CD40. In another aspect,the APCs are dendritic cells.

In one embodiment, the heat shock protein component includes but is notlimited to HSP70 and/or an immune activating fragment and/or modifiedsequence thereof. In another aspect, the HSP70 or the immune activatingfragment and/or modified sequence thereof is from Mycobacteriumtuberculosis.

In one embodiment, this disclosure relates to a method for preparingactivated T cells, the method comprising:

a) providing activated antigen-presenting cells that were activated inpresence of tumor cells and a fusion protein ex vivo for a time periodsufficient to produce activated antigen-presenting cells, wherein atleast one activated antigen-presenting cell displays an antigen derivedfrom the tumor cells;

b) contacting the activated antigen-presenting cells with T cells for aperiod of time sufficient to activate the T cells; and

c) isolating the activated T cells; wherein the fusion protein comprisesa tumor binding component and a heat shock protein component, whereinsaid tumor binding component binds to the tumor cells and said heatshock protein component activates antigen-presenting cells. In oneembodiment, the isolated activated T cells are free or substantiallyfree of the dendritic cells, tumor cells and the fusion protein.

In one embodiment, the heat shock protein component comprises HSP70 oran immune activating fragment and/or modified sequence thereof. In oneembodiment, the HSP70 or the immune activating fragment and/or modifiedsequence thereof is from Mycobacterium tuberculosis. In one embodiment,the tumor binding component is a single chain antibody, a variabledomain, or a Fab domain.

In one embodiment, the APCs were expanded in the presence of a growthfactor. In one embodiment, the growth factor is a cytokine. In oneembodiment, the growth factor is selected from the group consisting ofFlt-3 ligand, GM-CSF, IL-4, M-CSF, IFNα, IL-1β, IL-4, IL-6, IL-13, IL-15and TNFα.

In one embodiment, the tumor cells are obtained from a patient. In oneembodiment, the tumor cells are derived from a patient to be treated asdescribed herein. In one embodiment, the tumor cells are derived from adifferent patient. In one embodiment, the tumor cells comprise a cancercell line.

In one embodiment, the term “tumor cells” that are incubated with theimmune cells and fusion protein refers to any tumor sample, includingbut not limited to intact cells, viable cells, non-viable cells,tumor-specific antigen (e.g., isolated antigen, partially isolatedantigen, recombinant antigen, etc.), and/or other cellular materialderived from a tumor or cancer cell or cell line. In some embodiments,the tumor sample comprises at least a subset of antigens that areidentical or similar to antigens associated with a patient's tumor,e.g., that will result in a T cell response against the patient's tumor.

In one embodiment, the isolated activated T cells are contacted with aneffective amount of an anti-chemorepellant agent. In one embodiment, theanti-chemorepellant agent is any of the agents disclosed previously,e.g., selected from the group consisting of AMD3100 or a derivativethereof, AMD11070 (also called AMD070), AMD12118, AMD11814, AMD13073,FAMD3465, C131, BKT140, CTCE-9908, KRH-2731, TC14012, KRH-3955,BMS-936564/MDX-1338, LY2510924, GSK812397, KRH-1636, T-20, T-22, T-140,TE-14011, T-14012, TN14003, TAK-779, AK602, SCH-351125, tannic acid, NSC651016, thalidomide, GF 109230X, an antibody that interferes withdimerization of a chemorepellant chemokine, and an antibody thatinterferes with dimerization of a receptor for a chemorepellantchemokine. In one embodiment, the anti-chemorepellant agent is AMD3100.In one embodiment, the amount of anti-chemorepellant agent is sufficientto bind to at least a subset of receptors, e.g., CXCR4 and/or CXCR7, onthe surface of the T cells.

In one embodiment, the APCs are allogeneic, autologous, or derived froma cell line. In one embodiment, the T cells are allogeneic, autologous,or derived from a cell line. In one embodiment, the APCs and/or the Tcells are isolated from a patient having cancer. In certain embodiments,the APCs and T cells are derived from the same source. e.g., the samepatient.

In one embodiment, either the APCs or the tumor cells are immobilized ona solid support. In one embodiment, the activated APCs are isolated byisolating the solid support from the tumor cells and fusion protein.

In one embodiment, the T cells are immobilized on a solid support. Inone embodiment, the activated T cells are isolated by isolating thesolid support from the APCs.

In one embodiment, the solid support comprises a surface of a column, asepharose bead, a gel, a matrix, a magnetic bead, or a plastic surface.

Methods of Treatment

The fusion protein as described herein can be used in combination withan anti-chemorepellant agent to treat a disease which is associated witha chemorepellant effect. In one embodiment, the disease is an abnormalcell or a tumor. In one embodiment, the disease is a cancer.

In one aspect, this invention relates to a method for treating a cancer,e.g., a tumor, in a patient wherein the cancer or tumor expresseschemorepellant properties, the method comprising administering to thepatient: (a) an effective amount of APCs (e.g., dendritic cells)prepared by incubating the APCs with cancer or tumor cells and a fusionprotein; and (b) concurrently administering to the patient an effectiveamount of an anti-chemorepellant agent; wherein the combination of theAPCs and the anti-chemorepellant agent treat the cancer or tumor. In oneembodiment, the fusion protein comprises a antigen (or target) bindingcomponent and a stress protein component, wherein the antigen (ortarget) binding component binds to the cancer or tumor and the stressprotein component activates APCs (e.g., dendritic cells), leading to thegeneration of CD3 positive T cells that target cancer antigens.

In one embodiment, the anti-chemorepellant agent and the APCs (e.g.,dendritic cells) are administered separately. In one embodiment, theanti-chemorepellant agent and the APCs (e.g., dendritic cells) areadministered simultaneously. In one embodiment, the anti-chemorepellantagent and the APCs (e.g., dendritic cells) are administeredsequentially.

In one embodiment, the anti-chemorepellant agent is administered priorto administration of the APCs (e.g., dendritic cells). In oneembodiment, the anti-chemorepellant agent is administered afteradministration of the APCs (e.g., dendritic cells). In one embodiment,the anti-chemorepellant agent is administered before, during and/orafter administration of the APCs (e.g., dendritic cells).

In some embodiments, the anti-chemorepellant agent is administeredbetween one minute and 24 hours prior to administration of the APCs(e.g., dendritic cells). In some embodiments, the anti-chemorepellantagent is administered 1, 2, 3, 4, 5, 6, or 7 days prior toadministration of the APCs (e.g., dendritic cells).

In some embodiments, the anti-chemorepellant agent is administered for aperiod of time sufficient to reduce or attenuate the chemorepellanteffect of the tumor, e.g., such that the anti-chemorepellant agent hasan anti-chemorepellant effect; the APCs (e.g., dendritic cells) can thenbe administered for a period of time during which the chemorepellanteffect of the tumor is reduced or attenuated.

In some embodiments, the anti-chemorepellant agent is administeredbetween one minute and 24 hours after administration of the APCs (e.g.,dendritic cells). In some embodiments, the anti-chemorepellant agent isadministered 1, 2, 3, 4, 5, 6, or 7 days after administration of theAPCs (e.g., dendritic cells).

In one embodiment, the anti-chemorepellant agent and/or the APCs (e.g.,dendritic cells) is administered intravenously, subcutaneously, orally,or intraperitoneally. In one embodiment, the anti-chemorepellant agentis administered proximal to (e.g., near or within the same body cavityas) the tumor. In one embodiment, the anti-chemorepellant agent isadministered directly into the tumor or into a blood vessel feeding thetumor. In one embodiment, the anti-chemorepellant agent is administeredsystemically. In a further embodiment, the anti-chemorepellant agent isadministered by microcatheter, an implanted device, or an implanteddosage form.

In one embodiment, the anti-chemorepellant agent is administered in acontinuous manner for a defined period. In another embodiment, theanti-chemorepellant agent is administered in a pulsatile manner. Forexample, the anti-chemorepellant agent may be administeredintermittently over a period of time.

Cancers or tumors that can be treated by the compounds and methodsdescribed herein include, but are not limited to: anal cancer, biliarytract cancer; brain cancer, including glioblastomas andmedulloblastomas; breast cancer; cervical cancer; choriocarcinoma; coloncancer; endometrial cancer; esophageal cancer, gastric cancer; head andneck cancer, hematological neoplasms, including acute lymphocytic andmyelogenous leukemia; multiple myeloma; AIDS associated leukemias andadult T cell leukemia lymphoma; intraepithelial neoplasms, includingBowen's disease and Paget's disease; liver cancer (hepatocarcinoma);lung cancer; lymphomas, including Hodgkin's disease and lymphocyticlymphomas; mesotheliomas, neuroblastomas; oral cancer, includingsquamous cell carcinoma; ovarian cancer, including those arising fromepithelial cells, stromal cells, germ cells and mesenchymal cells;pancreas cancer; prostate cancer; rectal cancer; sarcomas, includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma andosteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma,basocellular cancer and squamous cell cancer; testicular cancer,including germinal tumors (seminoma, non-seminoma[teratomas,choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer,including thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor. In importantembodiments, cancers or tumors escaping immune recognition includeglioma, colon carcinoma, colorectal cancer, anal cancer, head and neckcancer, ovarian cancer, lymphoid cell-derived leukemia, choriocarcinoma,and melanoma. In one embodiment, the tumor cells derive frommesothelioma. In one embodiment, the tumor cells are from ahematological malignancy. In some embodiments, the cancer is aHPV-positive cancer.

In one embodiment, the term “tumor cells” refers to a tumor sample,including but not limited to intact cells, viable cells, non-viablecells, tumor-specific antigen (e.g., isolated antigen, partiallyisolated antigen, recombinant antigen, etc.), and/or other cellularmaterial derived from a tumor or cancer cell.

In one embodiment, the cancer is a solid tumor. In one embodiment, thecancer is a leukemia. In one embodiment, the cancer over-expressesCXCL12, e.g., expresses an amount of CXCL12 in the tumormicroenvironment sufficient to have a chemorepellant effect. In oneembodiment, cancer expression of CXCL12 can be evaluated prior toadministration of a composition as described herein. For example, apatient having a cancer that is determined to express or over-expressCXCL12, e.g., a concentration of about 100 nM or higher, e.g., about100, 150, 200, 250, 300, 350, 400, 450, or 500 nM, will be treated usinga method and/or composition as described herein.

In one embodiment, the patient is also administered an effective amountof a fusion protein. The fusion protein may be administered before, atapproximately the same time as, or after administration of the activatedAPCs and/or anti-chemorepellant agent. Dosages and routes ofadministration of the fusion protein are provided, for example in U.S.Pat. Nos. 8,143,387 and 7,943,133; U.S. Patent Pub. No. 20110129484; andU.S. Provisional Application No. 62/306,168; each of which isincorporated herein by reference in its entirety.

The activated T cells as described herein can be used to treat a cancer,cancer cell, or tumor in a patient that expresses an antigen recognizedby the activated T cells.

The activated T cells as described herein can be used in combinationwith an anti chemorepellant agent to treat a cancer, cancer cell, ortumor which is associated with a chemorepellant effect. For example, thecancer cell, cancer, or tumor expresses an amount of a chemorepellantcytokine, e.g., CXCL12, e.g., at a level of about 100 nM or more, thatis sufficient to repel or otherwise prevent access of immune cells tothe cancer.

In one aspect, this invention relates to a method for treating a cancer,e.g., a tumor, in a patient, the method comprising administering to thepatient: (a) an effective amount of APCs (e.g., dendritic cells)prepared by incubating the APCs with cancer or tumor cells and a fusionprotein; and (b) concurrently administering to the patient an effectiveamount of an anti-chemorepellant agent; wherein the combination of theAPCs and the anti-chemorepellant agent treat the cancer or tumor. In oneembodiment, the fusion protein comprises an antigen-binding componentand a stress protein component, wherein the antigen-binding componentbinds to the cancer or tumor and the stress protein component activatesAPCs (e.g., dendritic cells).

In one embodiment, the method comprises selecting a patient having atumor that exhibits a chemorepellant effect. In one embodiment, thetumor overexpresses CXCL12.

In one embodiment, the anti-chemorepellant agent and the activated Tcells are administered separately, e.g., by the same or differentroutes. In one embodiment, the anti-chemorepellant agent and theactivated T cells are administered simultaneously, in the same ordifferent compositions, e.g., by the same or different routes. In oneembodiment, the anti-chemorepellant agent and the activated T cells areadministered sequentially, e.g., by the same or different routes.

In one embodiment, the anti-chemorepellant agent is administered priorto administration of the activated T cells. In one embodiment, theanti-chemorepellant agent is administered after administration of theactivated T cells. In one embodiment, the anti-chemorepellant agent isadministered before, during and/or after administration of the activatedT cells.

In some embodiments, the anti-chemorepellant agent is administeredbetween one minute and 24 hours prior to administration of the activatedT cells. In some embodiments, the anti-chemorepellant agent isadministered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of theactivated T cells.

In some embodiments, the anti-chemorepellant agent is administered for aperiod of time sufficient to reduce or attenuate the chemorepellanteffect of the tumor, e.g., such that the anti-chemorepellant agent hasan anti-chemorepellant effect; the activated T cells can then beadministered for a period of time during which the chemorepellant effectof the tumor is reduced or attenuated.

In some embodiments, the anti-chemorepellant agent is administeredbetween one minute and 24 hours after administration of the activated Tcells. In some embodiments, the anti-chemorepellant agent isadministered 1, 2, 3, 4, 5, 6, or 7 days after administration of theactivated T cells.

In one embodiment, the anti-chemorepellant agent and/or the activated Tcells is administered intravenously, subcutaneously, orally, orintraperitoneally. In one embodiment, the anti-chemorepellant agent isadministered proximal to (e.g., near or within the same body cavity as)the tumor. In one embodiment, the anti-chemorepellant agent isadministered directly into the tumor or into a blood vessel feeding thetumor. In one embodiment, the anti-chemorepellant agent is administeredsystemically. In a further embodiment, the anti-chemorepellant agent isadministered by microcatheter, an implanted device, or an implanteddosage form.

In one embodiment, the anti-chemorepellant agent is administered in acontinuous manner for a defined period. In another embodiment, theanti-chemorepellant agent is administered in a pulsatile manner. Forexample, the anti-chemorepellant agent may be administeredintermittently over a period of time.

Cancers or tumors that can be treated by the compounds and methodsdescribed herein include, but are not limited to: anal cancer, biliarytract cancer; brain cancer, including glioblastomas andmedulloblastomas; breast cancer; cervical cancer; choriocarcinoma; coloncancer; endometrial cancer; esophageal cancer, gastric cancer; head andneck cancer, hematological neoplasms, including acute lymphocytic andmyelogenous leukemia; multiple myeloma; AIDS associated leukemias andadult T cell leukemia lymphoma; intraepithelial neoplasms, includingBowen's disease and Paget's disease; liver cancer (hepatocarcinoma);lung cancer; lymphomas, including Hodgkin's disease and lymphocyticlymphomas; mesothelioma, neuroblastomas; oral cancer, including squamouscell carcinoma; ovarian cancer, including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreas cancer;prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma,rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skincancer, including melanoma, Kaposi's sarcoma, basocellular cancer andsquamous cell cancer; testicular cancer, including germinal tumors(seminoma, non-seminoma[teratomas, choriocarcinomas]), stromal tumorsand germ cell tumors; thyroid cancer, including thyroid adenocarcinomaand medullar carcinoma; and renal cancer including adenocarcinoma andWilms tumor. In important embodiments, cancers or tumors escaping immunerecognition include glioma, colon carcinoma, colorectal cancer, analcancer, head and neck cancer, ovarian cancer, lymphoid cell-derivedleukemia, choriocarcinoma, and melanoma. In one embodiment, the tumorcells derive from mesothelioma. In one embodiment, the tumor cells arefrom a hematological malignancy. In one embodiment, the cancer isHPV-positive.

In one embodiment, the cancer is a solid tumor. In one embodiment, thecancer is a leukemia. In one embodiment, the caner over-expressesCXCL12. In one embodiment, cancer expression of CXCL12 can be evaluatedprior to administration of a composition as described herein. Forexample, a patient having a cancer that is determined to express orover-express CXCL12 will be treated using a method and/or composition asdescribed herein.

In one embodiment, the patient is also administered an effective amountof a fusion protein. The fusion protein may be administered before, atapproximately the same time as, or after administration of the activatedT cells and/or anti-chemorepellant agent. Dosages and routes ofadministration of the fusion protein are provided, for example in U.S.Pat. Nos. 8,143,387 and 7,943,133; U.S. Patent Pub. No. 20110129484; andPCT Application No. PCT/US2017/021911; each of which is incorporatedherein by reference in its entirety.

Dose and Administration

The compositions, as described herein, are administered in effectiveamounts. The effective amount will depend upon the mode ofadministration, the particular condition being treated and the desiredoutcome. It will also depend upon, as discussed above, the stage of thecondition, the age and physical condition of the subject, the nature ofconcurrent therapy, if any, and like factors well known to the medicalpractitioner. For therapeutic applications, it is that amount sufficientto achieve a medically desirable result.

Generally, the dose of the anti-chemorepellant agent of the presentinvention is from about 0.001 to about 100 mg/kg body weight per day,e.g., about 5 mg/kg body weight per day to about 50 mg/kg per day, e.g.,about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/kg per day inclusiveof all values and ranges therebetween, including endpoints. In oneembodiment, the dose is from about 10 mg/kg to about 50 mg/kg per day.In one embodiment, the dose is from about 10 mg/kg to about 40 mg/kg perday. In one embodiment, the dose is from about 10 mg/kg to about 30mg/kg per day. In one embodiment, the dose is from about 10 mg/kg toabout 20 mg/kg per day. In one embodiment, the dose does not exceedabout 50 mg/kg per day.

In one embodiment, the dose of the anti-chemorepellant agent is fromabout 50 mg/kg per week to about 350 mg/kg per week, inclusive of allvalues and ranges therebetween, including endpoints. In one embodiment,the dose of the anti-chemorepellant agent is about 50 mg/kg per week. Inone embodiment, the dose of the anti-chemorepellant agent is about 60mg/kg per week. In one embodiment, the dose of the anti-chemorepellantagent is about 70 mg/kg per week. In one embodiment, the dose of theanti-chemorepellant agent is about 80 mg/kg per week. In one embodiment,the dose of the anti-chemorepellant agent is about 90 mg/kg per week. Inone embodiment, the dose of the anti-chemorepellant agent is about 100mg/kg per week. In one embodiment, the dose of the anti-chemorepellantagent is about 110 mg/kg per week. In one embodiment, the dose of theanti-chemorepellant agent is about 120 mg/kg per week. In oneembodiment, the dose of the anti-chemorepellant agent is about 130 mg/kgper week. In one embodiment, the dose of the anti-chemorepellant agentis about 140 mg/kg per week. In one embodiment, the dose of theanti-chemorepellant agent is about 150 mg/kg per week. In oneembodiment, the dose of the anti-chemorepellant agent is about 160 mg/kgper week. In one embodiment, the dose of the anti-chemorepellant agentis about 170 mg/kg per week. In one embodiment, the dose of theanti-chemorepellant agent is about 180 mg/kg per week. In oneembodiment, the dose of the anti-chemorepellant agent is about 190 mg/kgper week. In one embodiment, the dose of the anti-chemorepellant agentis about 200 mg/kg per week. In one embodiment, the dose of theanti-chemorepellant agent is about 210 mg/kg per week. In oneembodiment, the dose of the anti-chemorepellant agent is about 220 mg/kgper week. In one embodiment, the dose of the anti-chemorepellant agentis about 230 mg/kg per week. In one embodiment, the dose of theanti-chemorepellant agent is about 240 mg/kg per week. In oneembodiment, the dose of the anti-chemorepellant agent is about 250 mg/kgper week. In one embodiment, the dose of the anti-chemorepellant agentis about 260 mg/kg per week. In one embodiment, the dose of theanti-chemorepellant agent is about 270 mg/kg per week. In oneembodiment, the dose of the anti-chemorepellant agent is about 280 mg/kgper week. In one embodiment, the dose of the anti-chemorepellant agentis about 290 mg/kg per week. In one embodiment, the dose of theanti-chemorepellant agent is about 300 mg/kg per week. In oneembodiment, the dose of the anti-chemorepellant agent is about 310 mg/kgper week. In one embodiment, the dose of the anti-chemorepellant agentis about 320 mg/kg per week. In one embodiment, the dose of theanti-chemorepellant agent is about 330 mg/kg per week. In oneembodiment, the dose of the anti-chemorepellant agent is about 340 mg/kgper week. In one embodiment, the dose of the anti-chemorepellant agentis about 350 mg/kg per week.

In one aspect of the invention, administration of theanti-chemorepellant agent is pulsatile. In one embodiment, an amount ofanti-chemorepellant agent is administered every 1 hour to every 24hours, for example every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, or 24 hours. In one embodiment, anamount of anti-chemorepellant agent is administered every 1 day, 2 days,3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

In one aspect of the invention, doses of the anti-chemorepellant agentare administered in a pulsatile manner for a period of time sufficientto have an anti-chemorepellant effect (e.g., to attenuate thechemorepellant effect of the tumor cell). In one embodiment, the periodof time is between about 1 day and about 10 days. For example, theperiod of time may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, or 10 days. In some embodiments, the APCs or Tcells are administered after the anti-chemorepellant effect hasoccurred, e.g., one or more days after the anti-chemorepellant agent hasbeen administered. In some embodiments, administration of thechemorepellant agent is continued while the APCs or T cells are beingadministered.

A variety of administration routes are available. The methods of theinvention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects.

Modes of administration include oral, rectal, topical, nasal,intradermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes are not particularly suitable for long-term therapyand prophylaxis. They could, however, be preferred in emergencysituations. Oral administration will be preferred for prophylactictreatment because of the convenience to the patient as well as thedosing schedule. When peptides are used therapeutically, in certainembodiments a desirable route of administration is by pulmonary aerosol.Techniques for preparing aerosol delivery systems containing peptidesare well known to those of skill in the art. Generally, such systemsshould utilize components which will not significantly impair thebiological properties of the antibodies, such as the paratope bindingcapacity (see, for example, Sciarra and Cutie, “Aerosols,” inRemington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712;incorporated by reference). Those of skill in the art can readilydetermine the various parameters and conditions for producing antibodyor peptide aerosols without resort to undue experimentation.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent(s). Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed 25oils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

In one embodiment, the anti-chemorepellant agent is administeredparenterally. In one embodiment, the anti-chemorepellant agent isadministered via microcatheter into a blood vessel proximal to a tumor.In one embodiment, the anti-chemorepellant agent is administered viamicrocatheter into a blood vessel within a tumor. In one embodiment, theanti-chemorepellant agent is administered subcutaneously. In oneembodiment, the anti-chemorepellant agent is administered intradermally.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the anti-chemorepellant agent, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono-, di-, andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like.

In one embodiment, the anti-chemorepellant agent or the APCs (e.g.,dendritic cells) or activated T cells is administered in a time-release,delayed release or sustained release delivery system. In one embodiment,the time-release, delayed release or sustained release delivery systemcomprising the anti-chemorepellant agent or the APCs (e.g., dendriticcells) or activated T cells is inserted directly into the tumor. In oneembodiment, the time-release, delayed release or sustained releasedelivery system comprising the anti-chemorepellant agent or the APCs(e.g., dendritic cells) or activated T cells is implanted in the patientproximal to the tumor. Additional implantable formulations aredescribed, for example, in U.S. Patent App. Pub. No. 2008/0300165, whichis incorporated herein by reference in its entirety.

In addition, important embodiments of the invention include pump-basedhardware delivery systems, some of which are adapted for implantation.Such implantable pumps include controlled-release microchips. Anexemplary controlled-release microchip is described in Santini, J T Jr.et al., Nature, 1999, 397:335-338, the contents of which are expresslyincorporated herein by reference.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

Pharmaceutical Compositions

In one aspect, this invention relates to a pharmaceutical compositioncomprising an anti-chemorepellant agent and APCs (e.g., dendritic cells)or activated T cells as described herein. In one embodiment, the APCs(e.g., dendritic cells) or T cells are derived from the patient to betreated. In one embodiment, the APCs (e.g., dendritic cells) or T cellsexpress CXCR4 and/or CXCR7 on the cell surface.

In one embodiment, the composition further comprises a fusion protein asdescribed herein.

In one embodiment, the composition comprises an effective amount of thefusion protein to activate the antigen-presenting cells. In oneembodiment, the composition comprises an effective amount of theanti-chemorepellant agent to reduce the chemorepellant effect of thecancer. In one embodiment, the composition comprises an effective amountof the antigen presenting cells to result in activation of T cellsagainst the cancer.

In one embodiment, the pharmaceutical composition is formulated forinjection.

In one embodiment, the composition comprises a pharmaceuticallyacceptable excipient. Acceptable excipients are non-toxic, aidadministration, and do not adversely affect the therapeutic benefit ofthe claimed compounds. Such excipient may be any solid, liquid,semi-solid or gaseous excipient that is generally available to one ofskill in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols. Othersuitable pharmaceutical excipients and their formulations are describedin Remington's Pharmaceutical Sciences, edited by E. W. Martin (MackPublishing Company, 22nd ed., 2013).

In order to ensure that the dendritic cells and fusion protein interactonce administered to a patient, the cells and protein may be combinedprior to administration. In one embodiment, the cells and protein arecombined immediately (e.g., seconds to one or two hours) prior toadministration to the patient.

Without being bound by theory, it is believed that combination of theAPCs or T cells and the anti-chemorepellant agent prior toadministration to the patient will allow binding of theanti-chemorepellant agent to cell surface receptors (e.g., CXCR4 and/orCXCR7), thereby allowing the cells to overcome the chemorepellant effectof the tumor. In contrast, it is contemplated that the systemic deliveryof an anti-chemorepellant agent results in indiscriminate binding ofthat agent to CXCR4 receptors and/or CXCR7 throughout the body.

In one embodiment, this invention relates to an ex vivo device orcontainer comprising activated APCs and T cells as described herein. Thedevice or container is preferably capable of being maintained undercontrolled, reproducible conditions such that the activated APCs and Tcells can interact to activate the T cells. For example, the device orcontainer may be, without limitation, a cell culture dish or plate, abag (e.g., a culture bag), or any other device or container that issuitable for growing and/or maintaining cells; a tube or column; abioreactor; etc.

Kit of Parts

In one aspect, this invention relates to a kit of parts for treatment ofa disease, the kit comprising a fusion protein and ananti-chemorepellant agent.

In one embodiment is provided a kit of parts for treatment of a cancer,e.g., a tumor, in a patient, the kit comprising a therapeuticallyeffective amount of an anti-chemorepellant agent and/or the APCs (e.g.,dendritic cells) or activated T cells prepared by the methods describedherein.

In one embodiment, the anti-chemorepellant agent is selected from thegroup consisting of AMD3100 or a derivative thereof, AMD11070 (alsocalled AMD070), AMD12118, AMD11814, AMD13073, FAMD3465, C131, BKT140,CTCE-9908, KRH-2731, TC14012, KRH-3955, BMS-936564/MDX-1338, LY2510924,GSK812397, KRH-1636, T-20, T-22, T-140, TE-14011, T-14012, TN14003,TAK-779, AK602, SCH-351125, tannic acid, NSC 651016, thalidomide, GF109230X, an antibody that interferes with dimerization of achemorepellant chemokine, and an antibody that interferes withdimerization of a receptor for a chemorepellant chemokine. In oneembodiment, the anti-chemorepellant agent is AMD3100 or a derivativethereof. In one embodiment, the anti-chemorepellant agent is AMD3100.

In one embodiment, the activated T cells and/or the anti-chemorepellantagent are formulated for injection.

In one embodiment, the activated T cells and the anti-chemorepellantagent are in the same formulation.

In one embodiment, the kit further comprises instructions for treatingthe cancer or tumor. In one embodiment, the kit of parts comprisesinstructions in a readable medium for dosing and/or administration ofthe anti-chemorepellant agent and the activated T cells.

The term “readable medium” as used herein refers to a representation ofdata that can be read, for example, by a human or by a machine.Non-limiting examples of human-readable formats include pamphlets,inserts, or other written forms. Non-limiting examples ofmachine-readable formats include any mechanism that provides (i.e.,stores and/or transmits) information in a form readable by a machine(e.g., a computer, tablet, and/or smartphone). For example, amachine-readable medium includes read-only memory (“ROM”); random accessmemory (“RAM”); magnetic disk storage media; optical storage media; andflash memory devices. In one embodiment, the machine-readable medium isa CD-ROM. In one embodiment, the machine-readable medium is a USB drive.In one embodiment, the machine-readable medium is a Quick Response Code(“QR Code”) or other matrix barcode.

EXAMPLES Example 1. Construction of VIC-008 Fusion Protein

The fusion protein scFv-MtbHsp70 was constructed with VH and VL fromanti-MSLN p4 scFv (Bergan L, et al. Development and in vitro validationof anti-mesothelin biobodies that prevent CA125/Mesothelin-dependentcell attachment. Cancer Letters. 2007; 255(2):263-74) fused to fulllength MtbHsp70 with a (G4S)3 linker in between, which has been shown inour previous study (Yuan J, et al. A novel mycobacterialHsp70-containing fusion protein targeting mesothelin augments antitumorimmunity and prolongs survival in murine models of ovarian cancer andmesothelioma. Journal of Hematology & Oncology. 2014; 7:15). Theoriginal fusion protein VIC-007, on which VIC-008 was based, achievedsignificant control of tumor growth and prolongation of the survival oftumor-bearing mice, but the antitumoral efficacy of the treatmentregimen was not optimal. Antigenic peptides bind to MtbHsp70 throughnon-covalent interactions and can elicit both MHC class I-restrictedCD8+ and MHC class II-restricted CD4+ T-cell responses. A new version ofthe scFv-MtbHsp70 fusion protein, VIC-008, was developed, which wasmodified from the original VIC-007 by the elimination of redundant aminoacids and the introduction of a single amino acid mutation,phenylalanine (F) in place of valine (V), at position 381 of MtbHsp70(FIG. 1). This change is designed to prevent peptide binding whileretaining the immune-stimulatory capacity of the protein, in order toreduce the possibility that MtbHsp70 might incidentally bind and deliverother antigens that could result in off target effects or the inductionof tolerance or autoimmunity.

The fusion proteins were constructed and expressed by WuXi App Tech(Shanghai, China) in CHO cells and provided at a purity of above 95% byHPLC and an endotoxin level of less than 1.0 EU/mg. VIC-008 is furtherdescribed in Zeng Y., et al. (Improved Antitumoral Efficacy ofMesothelin Targeted Immune Activating Fusion Protein in Murine Model ofOvarian Cancer. Int J Cancer Clin Res 2016; 3:05) and U.S. ProvisionalApplication No. 62/306,168, each of which is incorporated herein byreference in its entirety.

Example 2. VIC-008 Enhances Control of Tumor Growth

Ovarian cancer was established by intraperitoneal (i.p.) injection ofsyngeneic cancer cells, Luc-ID8 (5×10⁶ cells per mouse), into 6-week-oldfemale C57BL/6 mice. All mice were purchased from Jackson laboratories.Mice were observed daily for 1 week after inoculation of tumor cells.Tumor generations were consistently first evident via abdominaldistension secondary to malignant ascites, and tumor-bearing mice wereeuthanized at the endpoint when there were signs of distress, includingfur ruffling, rapid respiratory rate, hunched posture, reduced activity,and progressive ascites formation.

Mice with ovarian tumors were treated 7 days after tumor cellinoculation with i.p. injections of VIC-007 (4 μg per mouse), VIC-008 (4μg per mouse), or normal saline. This was followed by 3 furthertreatments at 7-day intervals.

Intraperitoneal tumor growth was monitored weekly after tumor cellinoculation using in vivo live imaging by IVIS Spectrum (PerkinElmer).Mice were injected intraperitoneally with 150 mg/kg body weight ofD-luciferin 10 min in advance and subsequently imaged by IVIS Spectrum.

As shown in FIG. 2, both VIC-007 and VIC-008 significantly slowed tumorgrowth as recorded by bioluminescence signals compared to saline(p<0.0001 and p<0.0001) while VIC-008 further significantly delayedtumor growth compared to VIC-007 (p<0.0001). Statistical differencesbetween three or more experimental groups were analyzed using Two-WayANOVA, followed by Tukey's multiple comparison tests when mean of eachgroup is compared with that of every other group.

Example 3. VIC-008 Enhances the Prolongation of Mouse Survival

The efficacy of VIC-007 and VIC-008 to prolong survival in thetumor-bearing mice was evaluated in the mice treated as described inExample 2.

As shown in FIG. 3, both VIC-007 and VIC-008 significantly enhanced thesurvival of tumor-bearing mice compared to saline (p=0.0253 andp=0.0002), with increased median survival of 55 days from saline to 60days from VIC-007 and further to 65 days from VIC-008. VIC-008 furthersignificantly prolonged the survival of the tumor-bearing mice comparedto VIC-007 (p=0.0301). Survival was analyzed with the Log-rank test.

Taken together, these data showed that the new version of the fusionprotein VIC-008 significantly delayed the tumor growth and prolonged thesurvival in a syngeneic murine model of ovarian cancer. Improved mousesurvival of VIC-008 compared to VIC-007 is likely related to the changesmade to the protein sequences.

Example 4. Combination Treatment of VIC-008 with AMD3100 FurtherProlongs Mouse Survival

Mesothelioma tumor was established by i.p. injection of cancer cells,Luc-40L or Luc-AE17, into C57BL/6 mice. One week later, saline, VIC-008(20 μg), and/or AMD3100 (1 μg/g body weight) were administered once aweek for four weeks, as indicated in FIG. 4.

As shown in FIG. 5 (Luc-40L), VIC-008 significantly enhanced thesurvival of tumor-bearing mice compared to saline (p=0.0214), while thecombination of VIC-008 and AMD3100 further enhanced survival (p<0.0001versus saline). Similar results were observed with Luc-AE17 (FIG. 6).AMD3100 had a slight effect on survival, but this effect did not reachsignificance in this study.

These data show that VIC-008 alone is able to prolong survival in bothmodels of mesothelioma, and AMD3100 alone slightly benefits survival.Combination treatment with VIC-008 and AMD3100 significantly suppressesmesothelioma proliferation and prolongs survival of mice withmesothelioma tumors.

Example 5. Combination Treatment of VIC-008 with AMD3100 ForMesothelioma

Materials and Methods

Reagents

The fusion proteins were constructed as described above and expressed byWuXi Biologics (Shanghai, China) in CHO cells and provided at a purityof above 95% by HPLC and an endotoxin level of less than 1.0 EU/mg.AMD3100 was purchased from Abcam (#ab120718).

Tumor Cells

40L and AE17 mouse mesothelioma cell lines were kind gifts from Dr.Agnes Kane in Department of Pathology and Laboratory Medicine at BrownUniversity. Cells were cultured at 37° C. in DMEM supplemented with 1%L-glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum.

Animal Models

Five-week-old female C57BL/6 mice were obtained from the JacksonLaboratory and maintained in the gnotobiotic animal facility ofMassachusetts General Hospital (MGH) in compliance with institutionalguidelines and policies. After one week acclimatization, tumors wereinitiated with 4×10⁶ 40L cells or 2×10⁶ AE17 cells per mouseadministered intraperitoneally (i.p.). A subset of the mice from eachgroup were euthanized with i.p. administration of Ketamine (9 mg/ml insaline) and Xylazine (0.9 mg/ml in saline) 7 days after the lasttreatment, and samples harvested for immune profiling of tumors, lymphnodes and spleens. The remaining animals in each group were monitoredfor survival. For survival studies, the mice were observed daily afterinoculation of tumor cells. Tumor generation was consistently firstevident via the appearance of abdominal distension secondary tomalignant ascites, and tumor-bearing mice were euthanized at theendpoint when there were signs of distress, including fur ruffling,rapid respiratory rate, hunched posture, reduced activity, andprogressive ascites formation.

Splenocytes from transgenic T-Red/FoxP3 GFP mice were used as a sourceof fluorescently tagged T_(g) cells by cell sorting as described below.T-Red/FoxP3 GFP mice are a fully backcrossed C57BL/6 line of transgenicmice that produced by crossing T-red mice with FoxP3 GFP mice. T-redmice express dsRedII under the control of the CD4 promoter modified tolack the negative control element thereby allowing expression in bothCD4⁺ and CD8⁺ T cells. In FoxP3 GFP mice, GFP is expressed from undercontrol of the FoxP3 promoter with internal deletions to FoxP3 toprevent over expression. All animal studies were approved by theInstitutional Animal Care and Use Committee of MGH.

Treatment

Beginning seven days after tumor inoculation, treatments wereadministrated by i.p. injection once a week for four successive weeks.VIC-008 was administrated i.p. at 20 μg in 100 μl of saline per mouseonce a week and AMD3100 was given once a week by i.p. injection at 1mg/kg of body weight in 100 μl of saline.

Immune Profiling by Flow Cytometry

Tumors were mechanically disaggregated using sterile razor blades anddigested at 37° C. for 2 hours in RPMI 1640 with collagenase type IV for40L tumors or type I for AE17 tumors (2 mg/ml, Sigma), DNase (0.1 mg/ml,Sigma), hyaluronidase (0.1 mg/ml, Sigma), and BSA (2 mg/ml, Sigma). Cellsuspensions were passed through 100 μm filters to remove aggregates.Lymph nodes and spleens were mashed and filtered through 40 μmstrainers. Cells were washed with staining buffer (#420201, Biolegend)and stained with the conjugated antibodies for surface markers. Totallive cells were determined by LIVE/DEAD® staining (ThermoFisher,#L23105).

For intracellular cytokine detection, after staining of surface markers,cells were fixed and permeabilized with fixation/permeabilizationreagents from BioLegend (#424401) or eBioscience (#00-5521-00) andstained with the conjugated antibodies for intracellular markers.

Conjugated antibodies from eBioscience were as follows: CD40L (cloneMR1), and PD-1 (clone J43). The following conjugated antibodies werepurchased from BioLegend: CD3 (clone 17A2), CD4 (clone GK1.5), CD8a(clone 53-6.7), Foxp3 (clone MF-14), IL-2 (clone JES6-5H4), and IFN-γ(clone XMG1.2). CD25 (clone PC61) antibody was from BD Biosciences.

Flow cytometric analyses were performed using BD LSRFortessa X-20 (BDBiosciences). Gating strategies were determined by the FluorescenceMinus One. Flow data were analyzed by FlowJo V10 (TreeStar).

Ex-Vivo Culturing of Splenocytes and Cytokine Detection

The splenocytes were harvested from mashed spleens, filtered through 40μm cell strainers and treated with red blood cell lysis buffer. 2×10⁶splenocytes were placed per well in 24-well plates in RPMI 1640 mediumsupplemented with L-glutamine and stimulated with 2 μg/ml of recombinantmouse mesothelin (BioLegend, #594006) for 72 hours. Brefeldin A andMonesin (BioLegend, #420601 and #420701) were added into the culturemedium during the last five hours. Splenocytes were then harvested andintracellular cytokine staining performed using the correspondingantibodies for cytometric analysis.

In vitro Reprogramming of T_(reg) Cells

rGH-ffLuc-eGFP transgenic mice express Green Fluorescent Protein (GFP)in Foxp3⁺ T_(reg) cells. Spleens were collected from these mice andmashed and filtered through 40 μm strainers. Cells expressing GFP-Foxp3from CD4⁺ splenocytes were sorted on a FACSAria (BD Biosciences) andthen exposed to AMD3100 (5 μg/ml) in the presence or absence ofanti-CD3/CD28 antibody (1 μg/ml) for 24 hours. Brefeldin A and Monesin(BioLegend, #420601 and #420701) were added into the culture mediumduring the last five hours. The cells were then harvested and stainedwith the conjugated antibodies specific for CD3, CD4, CD25, Foxp3, IL-2and CD40L, and analyzed by flow cytometry.

Statistical Analyses

P values were calculated by GraphPad Prism 6. Unless describedotherwise, the P values for comparison among groups were obtained byOne-way ANOVA with Dunnett's multiple comparisons test or unpairedt-test with Welch's correction. The Kaplan-Meier method and log-ranktest were used to compare survival among groups. P<0.05 is consideredstatistically significant. Data are presented as mean±SEM.

Results

Combination Therapy with AMD3100 and VIC-008 Augments Tumor Control andMouse Survival

Two intraperitoneal malignant mesothelioma models were established inimmunocompetent C57BL/6 mice, separately using the syngeneic 40L andAE17 cell lines. Here, the effect of AMD3100 and VIC-008 was tested,used singly or in combination, on tumor growth and animal survival inmesothelioma-bearing mice. In animals treated with VIC-008 alone (20 μgper mouse), the total weight of intraperitoneal tumors collected oneweek after the last treatment was generally reduced (FIGS. 7A-7B) andanimal survival was significantly prolonged (FIGS. 7C-7D) compared tosaline control treatment in both the 40L and AE17 models (P<0.01 andP<0.01, respectively). AMD3100 alone at 1 mg/kg of mouse body weightconferred only modest benefit to survival in both 40L and AE17 mouse MMmodels compared to saline control treatment. However, the combinationtreatment with VIC-008 and AMD3100 significantly enhanced tumor control(P<0.0001 and P<0.001, respectively) and prolonged animal survival(P<0.0001 and P<0.0001, respectively) compared to saline control in both40L and AE17 models. Moreover, the combination treatment showed furthersignificantly improved antitumor efficacy on inhibition of tumor growth(P<0.001 and P<0.05, respectively) and prolongation of mouse survival(P<0.0001 and P<0.001, respectively) compared to VIC-008 monotherapy inboth 40L and AE17 models. These data indicate that when combined,AMD3100 significantly enhances the antitumor effect of VIC-008 in both40L and AE17 MM mouse models compared to monotherapy with either agent.

VIC-008 Increases Lymphocyte Infiltration in Spleens, Lymph Nodes andTumors

Spleens, axillary and inguinal lymph nodes, and intraperitoneal tumorswere collected from tumor-bearing mice one week after the lasttreatment. Single cells were prepared from these tissues and analyzed byflow cytometry (FIG. 8A). The proportions of CD8⁺ T cells in the totallive cells recovered from spleens (P<0.05 and P<0.05, respectively) andtumors (P<0.01 and P<0.01, respectively) for both the 40L and AE17models, and from lymph nodes (P<0.01) in AE17 model were significantlyincreased in the VIC-008 treatment group compared to that in the salinecontrol group (FIGS. 8B-8F). In VIC-008 and AMD3100 combinationtreatment group, the proportions of CD8⁺ T cells in the total live cellsrecovered from spleens (P<0.05 and P<0.05, respectively) and tumors(P<0.01 and P<0.05, respectively) for both the 40L and AE17 models, andfrom lymph nodes (P<0.001) in AE17 model were significantly increasedcompared to that in the saline control group. AMD3100 treatment did notincrease the proportion of CD8⁺ T cells in these tissues. Moreover,there was no difference in the proportion of CD8⁺ T cells between theVIC-008 and combination treatment groups, indicating that VIC-008increased lymphocyte infiltration of these tissues.

VIC-008 Enhances Tumor Antigen-Specific CD8⁺ T-Cell Responses

Next, single cells isolated from spleens and lymph nodes fromtumor-bearing mice were restimulated with recombinant mesothelin ex vivoand analyzed intracellular IFN-γ in CD8⁺ T cells. Mesothelin-specificIFN-γ expression in splenic CD8⁺ T cells both in 40L (P<0.001) and AE17(P<0.001) models, and in lymph node CD8⁺ T cells in the AE17 model(P<0.01), were significantly greater in mice treated with VIC-008 alonecompared to that in mice treated with saline (FIGS. 9A-9D). In VIC-008and AMD3100 combination treatment group, mesothelin-specific IFN-γexpression in splenic CD8⁺ T cells both in 40L (P<0.01) and AE17(P<0.01) models, and in lymph node CD8⁺ T cells in the AE17 model(P<0.001), were significantly greater compared to that in mice treatedwith saline. AMD3100 treatment by itself did not enhanceantigen-specific IFN-γ secretion in CD8⁺ T cells. Together, these datasupport the view that VIC-008 treatment enhances antitumor CD8⁺ T-cellresponses in both the 40L and AE17 mesothelioma mouse models.

AMD3100 Decreases PD-1 Expression on CD8⁺ T Cells

Next, expression of programmed cell death protein-1 (PD-1) on CD8⁺ Tcells was evaluated in spleen, tumor and lymph nodes. There was nosignificant difference between the VIC-008 treatment group and thesaline control group in the proportion of PD-1-expressing CD8⁺ T cellsin spleens in both the 40L and AE17 tumor-bearing mice, and in lymphnodes for AE17 mice (FIGS. 10A-10C). In marked contrast, significantlymore intratumoral CD8⁺ T cells in the VIC-008 treated group expressedPD-1 in the 40L tumors (P<0.05) and AE17 tumors (P<0.01) compared withthe saline-treated controls (FIGS. 10D-10E). The percentage of PD-1expressing CD8⁺ T cells ranged between 43-76% and 28-47% in the 40Ltumors and AE17 tumors respectively compared to only 5-10% in spleen andlymph nodes. These data indicate that the antitumor activity of the CD8⁺T cells in the tumor environment induced by VIC-008 treatment could beobstructed by activation of the PD-1/PD-L1 pathway.

Compared to this effect of VIC-008, and surprisingly, it was found thatAMD3100 reduced PD-1 expression on CD8⁺ T cells. AMD3100 treatment aloneled to significantly fewer PD-1-expressing CD8⁺ T cells in spleens(P<0.05 and P<0.01, respectively) and tumors (P<0.05 and P<0.05,respectively) in both the 40L and AE17 tumor-bearing mice, and in lymphnodes (P<0.01) for AE17 mice than in mice treated with saline. InAMD3100 and VIC-008 combination treatment group significantly fewer CD8⁺T cells expressed PD-1 in spleens (P<0.05 and P<0.01, respectively) andtumors (P<0.05 and P<0.05, respectively) in both the 40L and AE17tumor-bearing mice, and in lymph nodes (P<0.01) for AE17 mice comparedwith the saline-treated controls. There was no significant difference inthe proportion of PD-1-expressing CD8⁺ T cells in these tissues betweenthe AMD3100 monotherapy and AMD3100-VIC-008 combination therapy groups.These data indicated that AMD3100 could inhibit CD8⁺ T cells fromexpressing PD-1 in spleens, lymph nodes and tumors.

AMD3100 Reduces Tumor-Infiltrating T_(reg) Cells

Next, the impact of AMD3100 on T_(reg) cells was evaluated. AMD3100 didnot alter the proportions of T_(reg) cells found in spleens of 40Ltumor-bearing mice (FIGS. 11A-11B). In AE17 tumor-bearing mice AMD3100alone generally reduced T_(reg) in the lymph nodes (FIG. 11C) andAMD3100 alone or in combination with VIC-008 significantly increased thecell ratio of CD8⁺ T cells to T_(reg) cells (P<0.01 and P<0.0001,respectively) compared to saline treatment (FIG. 11D). In tumors fromboth the 40L and AE17 models, AMD3100 applied as monotherapysignificantly decreased the proportions of T_(reg) (P<0.05 and P<0.01,respectively) and increased the ratio of CD8⁺ T cells to T_(reg)(P<0.001 and P<0.01, respectively) compared to saline treatment (FIGS.11E-11F). In AMD3100 and VIC-008 combination treatment group theproportions of T_(reg) were significantly decreased (P<0.05 and P<0.05,respectively) and the ratio of CD8⁺ T cells to T_(reg) increased(P<0.001 and P<0.0001, respectively) compared to saline treatment inboth the 40L and AE17 models. In these two murine mesothelioma models,AMD3100 reduced intratumoral T_(reg) infiltration.

AMD3100 Modulates T_(reg) Cells Toward a T Helper Phenotype

It was observed that AMD3100, alone or in combination with VIC-008,significantly increased the ratio of CD25⁻ cells to CD25⁺ cells withinthe Foxp3⁺ population in both 40L (FIG. 12A, P<0.01 and P<0.001,respectively) and in the lymph nodes in the AE17 model (FIG. 12B,P<0.001 and P<0.01, respectively). Among the Foxp3⁺ CD25⁻ T_(reg)population significantly more cells were phenotypically IL-2⁺ CD40L⁺(FIGS. 12C-12E) after AMD3100 monotherapy and combination therapy withVIC-008, which suggested a change from T_(reg) cells to helper-likecells that had lost CD25 without loss of Foxp3, and may have lost theirimmunosuppressive function. There was no difference in the proportion ofIL-2⁺ CD40L⁺ cells in the Foxp3⁺ CD25⁻ T_(reg) population betweenAMD3100 monotherapy and combination therapy groups, indicating thatAMD3100 treatment may be the major driver of reprogramming of T_(reg)into helper-like cells.

AMD3100-Driven Modulation of T_(reg) Phenotype Requires TCR Activation

It was next addressed whether AMD3100-driven T_(reg) modulation could beinitiated in isolated single cells. Cells expressing GFP-Foxp3 from CD4⁺splenocytes in T-Red/FoxP3 GFP transgenic mice were sorted and treatedin vitro with AMD3100. AMD3100 treatment alone did not change the ratioof the proportion of CD25⁻ cells to CD25⁺ cells in the Foxp3⁺ CD4⁺population (FIGS. 13A-13B). However, in the presence of stimulation byanti-CD3/CD28 antibodies to trigger TCR activation, AMD3100 treatmentsignificantly increased the ratio of the proportion of CD25⁻ cells toCD25⁺ cells in the Foxp3⁺ CD4⁺ population (FIGS. 13C-13D, P=0.0017) andconverted more Foxp3⁺ CD25⁻ T_(reg) into IL-2⁺ CD40L⁺ cells (FIGS.13E-13F, P=0.0015). These data indicated that the conversion of T_(reg)cells into helper-like cells can be mediated by AMD3100 treatment ofsingle cells upon TCR activation.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

What is claimed is:
 1. A method for preparing activated antigen-presenting cells (APCs), the method comprising: a) incubating immune cells comprising APCs ex vivo in presence of cancer cells and a fusion protein for a time period sufficient to produce activated APCs, wherein at least one activated APC displays an antigen derived from the cancer cells; and b) isolating the activated APCs; wherein the fusion protein comprises an anti-MSLN scFv and a stress protein component, wherein said anti-MSLN scFv binds to mesothelin on one or more of the cancer cells and said stress protein component causes activation of the APCs, and wherein the method further comprises contacting the isolated activated APCs with an effective amount of an anti-chemorepellant agent, wherein the anti-chemorepellant agent is AMD3100.
 2. The method of claim 1, wherein the stress protein component comprises HSP70 and/or modified sequence thereof.
 3. The method of claim 2, wherein the stress protein component comprises Mycobacterium tuberculosis-derived HSP70 (MtbHsp70) and/or modified sequence thereof.
 4. The method of claim 1, wherein the cancer cells are derived from mesothelioma.
 5. The method of claim 1, further comprising expanding the APCs in presence of a growth factor.
 6. The method of claim 5, wherein the growth factor is a cytokine.
 7. The method of claim 6, wherein the growth factor is selected from a group consisting of Flt-3 ligand, GM-CSF, IL-4, M-CSF, IFNα, IL-1β, IL-4, IL-6, IL-13, IL-15 and TNFα.
 8. The method of claim 1, wherein the immune cells comprise dendritic cells, B lymphocytes, and/or mononuclear phagocytes, said mononuclear phagocytes comprising monocytes or macrophages.
 9. The method of claim 1, wherein at least one immune cell comprises a CD40 receptor.
 10. The method of claim 1, wherein the activated APCs comprise activated dendritic cells.
 11. The method of claim 10, wherein the dendritic cells are differentiated or matured from dendritic cell precursors.
 12. The method of claim 1, wherein the isolated activated APCs are free or substantially free of the cancer cells and/or the fusion protein.
 13. The method of claim 12, wherein the fusion protein comprises the peptide sequence of SEQ ID NO:
 2. 14. The method of claim 1, wherein the fusion protein comprises the peptide sequence of SEQ ID NO: 1 or SEQ ID NO:
 2. 